HEAT TOLERANCE AND ACCLIMATION IN FEMALE ATHLETES

HEATTOLERANCEANDACCLIMATIONINFEMALEATHLETES

JessicaAnneMee

ADoctoralthesissubmittedinfulfilmentoftherequirementoftheUniversityofBrighton

fortheawardofDoctorofPhilosophy

I

Abstract

Thisthesisaimedtodeveloparunningheattolerancetest(RHTT)toassesschangesinheattolerance

andtoinvestigatethemechanismandoptimisationofheatacclimation(HA)forfemaleathletes.

ThefirststudyintroducedaRHTTandassesseditsrepeatability.Resultsdemonstrategoodagreement,

strongcorrelationsandsmalldifferencesbetweenrepeatedtrials.Thetypicalerrorofmeasurevalues

suggested low within-participant variability. Furthermore, the RHTT was effective in differentiating

betweenindividuals’physiologicalresponses,demonstratingthatheattoleranceliesalongacontinuum.

ThesecondstudyexaminedthesensitivityoftheRHTTtochanges inheattoleranceandtoevaluate

individualresponsestoHA.ResultsdemonstratethattheRHTTissensitivetochangesinheattolerance

and that themagnitudeof adaptation is highly individual; supporting theuseof theRHTT in future

investigations.

ReducingthermalstrainthroughHAinnotfullyunderstoodforafemalepopulation.Thethirdstudy

comparedmales’ and females’ temporal patterning to short-termHA (STHA; 5-d) and long-termHA

(LTHA;10-d).TheRHTTwasusedtoquantifychangesinheattolerance.Theresultsconfirmthatwhilst

STHAmaybeeffectiveinachievingpartialadaptationinmalesandfemales,femalesrequireLTHAto

establishreductionsinthermoregulatoryandcardiovascularstrain.

ImprovedthermotolerancefollowingHA,reducesdisruptionstocellularhomeostasisprincipally,butnot

exclusively,byincreasingbasalheatshockprotein72followingtranscriptionofitsgene(Hsp72mRNA)

aspartoftheheatshockresponse(HSR).Thefourthstudycomparedmales’andfemales’Hsp72mRNA

responseduringSTHAandLTHA.ThesimilartranscriptionofHsp72mRNAobservedinallparticipants

suggeststhattherearenodifferencesintheendogenouscriteriatoelicittheHSRbetweensexes.

ThefifthstudyassessedtheeffectivenessofprecedingSTHAwithapassiveheatexposure(HAsauna)in

females.HAsaunaresultedinreductionsinthermoregulatory,cardiovascularandperceptualstrain.The

adaptation pathway was likely mediated in part, by plasma volume expansion and an improved

thermoeffectorandthermosensitivityresponseofthesudomotorfunction.

Together,evidenceinthisthesissupportsthenotionthatspecialconsiderationsneedtobetakenwhen

usingHAtoattenuatethermoregulatorystraininfemaleathletespriortotrainingandcompetinginthe

heat.

II

TableofContents

Abstract......................................................................................................................................I

Listofabbreviations................................................................................................................VIII

Listoftables...............................................................................................................................X

Listoffigures............................................................................................................................XII

Listofequations.......................................................................................................................XV

Acknowledgments...................................................................................................................XVI

Declaration.............................................................................................................................XVII

1. Introduction........................................................................................................................1

2. Literaturereview................................................................................................................6

2.1. Environmentalheatstressandexchange...........................................................................6

2.1.1. Dryheattransferpathways........................................................................................7

2.1.1. Wetheattransferpathways.......................................................................................8

2.1.2. Heatbalance...............................................................................................................8

2.2. Heatstrain........................................................................................................................10

2.2.1. Measurementofcoretemperature.........................................................................11

2.2.2. Measurementofskintemperature..........................................................................13

2.2.3. Bodyheatcontent....................................................................................................14

2.3. Behaviouraltemperatureregulation................................................................................17

2.4. Physiologicaltemperatureregulation..............................................................................17

2.4.1. Adjustablesetpoint.................................................................................................18

2.4.2. Reciprocalinhibition.................................................................................................20

2.4.3. Heatregulation.........................................................................................................21

2.5. Physiologicalresponsestoheatstress.............................................................................22

2.5.1. Sweatingandevaporativeheatloss.........................................................................23

III

2.5.2. Skinbloodflowanddryheatloss.............................................................................24

2.5.3. Cardiovascularsupportofthermoregulation...........................................................26

2.6. Sexdifferencesinphysiologicalresponsestoheatstress................................................27

2.6.1. Sweatrateandevaporativeheatloss......................................................................28

2.6.2. Skinbloodflowanddryheatloss.............................................................................28

2.7. Menstrualcycle................................................................................................................29

2.8. Verificationofthemenstrualcyclephase........................................................................32

2.8.1. Countingdaysfromtheonsetofmenses.................................................................32

2.8.2. Basalbodytemperature...........................................................................................33

2.8.3. Urinaryluteinisinghormoneconcentration.............................................................33

2.8.4. Measurementofoestrogenandprogesterone........................................................33

2.9. Thermoregulatorydifferencesoverthemenstrualcycle.................................................34

2.9.1. Sweatingandskinbloodflow...................................................................................35

2.9.2. Cardiovascular..........................................................................................................36

2.9.3. Exerciseperformanceintheheat............................................................................37

2.10. Individualdifferenceinheattolerance........................................................................38

2.10.1. Aerobicfitness..........................................................................................................38

2.10.2. Anthropometry.........................................................................................................40

2.11. Methodstoassessheattolerance................................................................................41

2.12. Exertionalheatillnessesinrunning..............................................................................42

2.13. Heatacclimation...........................................................................................................44

2.14. Heatacclimationprotocols...........................................................................................45

2.14.1. Fixedintensityheatacclimation...............................................................................45

2.14.2. Self-regulatedheatacclimation................................................................................46

2.14.3. Passiveheatacclimation..........................................................................................46

2.14.4. Controlledhyperthermiaheatacclimation..............................................................46

IV

2.15. Heatacclimationinduction..........................................................................................47

2.15.1. Sweatingandskinbloodflow...................................................................................47

2.15.2. Bloodvolumeandfluidbalance...............................................................................51

2.15.3. Cardiovascularstability............................................................................................54

2.15.4. Thermotolerance......................................................................................................58

2.15.5. Exerciseperformance...............................................................................................60

2.15.6. Perceptualresponses...............................................................................................62

2.16. Timecourseofheatacclimation..................................................................................62

2.17. Heatacclimationdecay................................................................................................63

2.18. Females’responsestoheatacclimation......................................................................64

2.19. Aimofthesis.................................................................................................................65

2.19.1. Researchquestionsarisingfromtheliteraturereview............................................65

2.19.2. Proposedresearchstudiesandhypotheses.............................................................66

3. Generalmethods..............................................................................................................68

3.1. Ethics,healthandsafety..................................................................................................68

3.2. Participants.......................................................................................................................69

3.3. Facilities............................................................................................................................70

3.4. Pre-trialdietandexercisestandardisation......................................................................70

3.5. Anthropometricassessment............................................................................................72

3.6. Menstrualcycle................................................................................................................74

3.7. Environmentalconditions................................................................................................75

3.8. Thermoregulatorymeasures............................................................................................76

3.9. Cardiopulmonarymeasures.............................................................................................78

3.10. Physiologicalstrainindex.............................................................................................79

3.11. Haematologicalmeasures............................................................................................79

3.12. Sweatrate....................................................................................................................80

V

3.13. Perceptualscales..........................................................................................................81

3.14. Cyclingexercisetrials...................................................................................................82

3.15. Runningexercisetrials..................................................................................................85

3.16. Heattolerancetests.....................................................................................................86

3.17. Statisticalanalysis.........................................................................................................91

3.17.1. Poweranalysis..........................................................................................................91

3.17.2. Normalityandsphericityofdata..............................................................................91

3.17.3. Repeatabilitystatistics..............................................................................................91

3.17.4. Statisticaltestsofdifference....................................................................................92

3.17.5. Effectsizes................................................................................................................93

3.17.6. Posthocanalysis......................................................................................................94

4. Repeatabilityofarunningheattolerancetest..................................................................95

4.1. Abstract............................................................................................................................95

4.2. Introduction......................................................................................................................95

4.3. Materialsandmethods....................................................................................................98

4.4. Results..............................................................................................................................99

4.5. Discussion.......................................................................................................................103

4.6. Limitations......................................................................................................................106

4.7. Conclusion......................................................................................................................106

5. Sensitivityofarunningheattolerancetest.....................................................................107

5.1. Abstract..........................................................................................................................107

5.2. Introduction....................................................................................................................107

5.3. Materialsandmethods..................................................................................................109

5.4. Results............................................................................................................................111

5.5. Discussion.......................................................................................................................114

5.6. Limitations......................................................................................................................116

VI

5.7. Conclusion......................................................................................................................117

6. Acomparisonofmaleandfemaletemporalpatterningtoshort-andlong-termheatacclimation.............................................................................................................................118

6.1. Abstract..........................................................................................................................118

6.2. Introduction....................................................................................................................118


6.4. Results............................................................................................................................123

6.5. Discussion.......................................................................................................................131

6.6. Limitations......................................................................................................................134

6.7. Conclusion......................................................................................................................135

7. SexcomparisonofleukocyteHsp72mRNAtranscriptionduringheatacclimation...........136

7.1. Abstract..........................................................................................................................136

7.2. Introduction....................................................................................................................136


7.4. Results............................................................................................................................141

7.5. Discussion.......................................................................................................................146

7.6. Limitations......................................................................................................................147

7.7. Conclusion......................................................................................................................148

8. Passiveheatexposureprecedingshort-termheatacclimationacceleratesadaptationinfemales..................................................................................................................................149

8.1. Abstract..........................................................................................................................149

8.2. Introduction....................................................................................................................149


8.4. Results............................................................................................................................155

8.5. Discussion.......................................................................................................................163

8.6. Limitations......................................................................................................................165

8.7. Conclusion......................................................................................................................165

VII

9. Generaldiscussion..........................................................................................................167

9.1. PrincipleFindings............................................................................................................167

9.1.1. Heattolerancetest.................................................................................................167

9.1.2. Sexcomparisonstoheatacclimation.....................................................................167

9.1.3. Acceleratedheatacclimationresponsesinfemales..............................................168

9.2. MechanisticOverview....................................................................................................171

9.2.1. HeatTolerance.......................................................................................................171

9.2.2. HeatAcclimation....................................................................................................176

9.2.3. Acceleratedheatacclimationinfemales................................................................181

9.3. Directionsforfutureresearch........................................................................................182

9.4. Practicalapplicationoffindings.....................................................................................185

9.5. Conclusion......................................................................................................................186

References.............................................................................................................................188

Appendices............................................................................................................................201

Informedconsent.......................................................................................................................201

Medicalquestionnaire...............................................................................................................202

Menstrualcyclequestionnaire...................................................................................................204

Calculationof65%VbO2peak......................................................................................................205

VIII

Listofabbreviations

BM Bodymass

BSA Bodysurfacearea

CI Confidenceinterval

CO2 Carbondioxide

FSH Folliclestimulatinghormone

HA Heatacclimation

HAsauna HAcombinedwithapassiveheatexposure

HR Heartrate

HspmRNA Heatshockproteinmessengerribonucleicacid

HSP Heatshockprotein

HSR Heatshockresponse

HTT Heattolerancetest

ICC Intraclasscorrelationcoefficient

IDF IsraeliDefenceForce

LH Luteinisinghormone

LTHA Long-termheatacclimation

NaCl SodiumChloride

O2 Oxygen

PV Plasmavolume

RH Relativehumidity

IX

RHTT Runningheattolerancetest

RPE Ratingofperceivedexertion

rpm revolutionspermin

RT-qPCR Reversetranscriptionpolymerasechainreaction

SD Standarddeviation

SWR Sweatrate

SWRBSA Sweatraterelativetobodysurfacearea

STHA Short-termheatacclimation

TEM Technicalerrorofmeasurement

TE(CV%) Technicalerrorasacoefficientofvariation

Tc Coretemperature

Tre Rectaltemperature

TS Thermalsensation

Tsk Meanskintemperature

USG Urinespecificgravity

VbO2max Maximaloxygenuptake

VbO2peak Peakoxygenuptake

WBGT Wetbulbglobetemperature

X

Listoftables

Table2.1Estimatedwholebodyskinbloodflowrequirementsduringprolonged,severerunning

exerciseatdifferentbodycoreandskintemperatures.

Table2.2Commonterminologyandhormoneconcentrationsassociatedwithatypical28-dcycle

withovulationoccurringonD14.

Table2.3Heatillnessesandtheirassociatedsymptoms.

Table2.4Summaryofadaptationstoheatacclimation.

Table2.5Change inwholebodysweat rateobserved following short-and long-termcontrolled

hyperthermiaheatacclimation.

Table 2.6 Change in plasma volume observed following short- and long-term controlled


Table 2.7 Change in resting heart rate observed following short- and long-term controlled


Table2.8Changeinrestingcoretemperatureobservedfollowingshort-andlong-termcontrolled


Table2.9Plateaudaysofphysiologicaladaptations(thepointatwhichapproximately85%ofthe

adaptationoccurs)duringheatacclimatisation.

Table3.1Repeatabilityofurineosmolalityandurinespecificgravitymeasurement.Mean±SD.

Table3.2Repeatabilityofskinfoldmeasurements.Mean±SD.

Table3.3Plasmaconcentrationof17β-estradiolandprogesterone.Mean±SD(range).

Table3.4Repeatabilityofrectalthermistors.Mean±SD.

Table3.5Repeatabilityofskinthermistors.Mean±SD.

Table3.6Repeatabilityofcapillarybloodlactateanalysis.Mean±SD.

Table3.7PhysiologicalandsubjectiveresponsetocyclingatsetpercentagesofVbO2peak.

XI

Table3.8PhysiologyvariablesmeasuredduringtheIDFHTTandtheRHTT.Mean±SD.

Table 4.1Repeatability statistics for physiological variables during repeated trials of the RHTT.

Mean±SD.

Table5.1Participantcharacteristics.Mean±SD(ranges).

Table5.2PeakphysiologicalvariablesduringtheRHTT.Mean±SD(ranges).

Table6.1Participantcharacteristics.Mean±SD.

Table6.2Performanceandphysiologicalresponsesduringshort-termheatacclimationandlong-

termheatacclimation.Mean±SD.

Table 6.3 Physiological variables during baseline testing (RHTT1), following STHA (RHTT2) and

followingLTHA(RHTT3).Mean±SD.


Table7.2Hsp72mRNAprimersequences.

Table7.3PhysiologicalandperformancemeasuresonD1,D5andD10ofcontrolledhyperthermia

heatacclimation.Mean±SD.

Table8.1Heat acclimationprotocol andphysiological responsesduringD1andD5ofHACH and

HAsauna.Mean±SD.

Table8.2Physiologicalvariablesduringbaselinetesting(RHTT1),andfollowing5-dHACHorHAsauna

(RHTT2).Mean±SD.

Table9.1Hypothesesforeachstudychapterpresentedinthisthesis.

Table9.2Participantcharacteristics.Mean±SD(Range).



Table9.4Daysofphysiologicaladaptation inmalesandfemalesduringcontrolledhyperthermia

heatacclimation.

XII

Listoffigures

Figure 2.1. Avenues for heat exchange when performing exercise. Adapted from Gisolfi and

Wenger,(1984)

Figure2.2Meanchangesincoretemperature(A)andmuscletemperature(B)duringandfollowing

60-minexercise.N=8(6M,2F)TakenfromKennyetal.,(2008).

Figure2.3Oesophageal,rectal,andintestinaltemperaturemeasuredsimultaneouslyduringrest,

submaximal supine cycling exercise at 40% VbO2 peak and 65% VbO2 peak, and during passive

recovery.N=7(5M,2F).

Figure2.4The2-compartmentthermometrymodelandthe3-compartmentthermometrymodel

forchangeinmeanbodytemperatureandchangeinbodyheatcontent.Adaptedfrom(Jayand

Kenny,2007;KennyandJay,2013).

Figure2.5Schematicdiagramofthethermoregulatorycontrolsystem.AdaptedfromSawkaetal.

(2011).

Figure2.6Thereciprocalinhibitionmodelofthermoregulation.Adaptedfrom(Bligh,2006).

Figure2.7Meanwholebodycalorimetrydataforrateoftotalheatproductionandrateofnetheat

loss after60-min rest, 60-min cyclingat70Wand60-minof inactive recovery.N=8 (6M,2F).

AdaptedfromKennyetal.(2008).

Figure2.8Typicalcoretemperature(Tc)andhormonechangesacrossthemenstrualcycle.Noteall

valuesareapproximatevaluessincetheyvarygreatlyacrossindividuals.LH=luteinizinghormone

FSH=folliclestimulatinghormone.AdaptedfromStachenfeld&Taylor,(2014).

Figure2.9Hsp72mRNApre-andpost-sessionsonD1,D5andD10ofcontrolledhyperthermiaHA

inmaleparticipants.Adaptedfrom(Gibsonetal.,2015a).

Figure2.10EffectofheatacclimationonVbO2maxinacool(13°C)andhot(38°C)environmentin

maleparticipants.

Figure3.1Ratingofperceivedexertion(RPE)andthermalsensation(TS)scales.

XIII

Figure3.2.Exampleofrelativeexerciseintensityelicitedduringa90-mincontrolledhyperthermia

heatacclimationsession.

Figure3.3Restingrectaltemperature(Tre)andrestingheartrate(HR)duringtheIsraeliDefence

Forceheattolerancetest(IDFHTT)(x-axis)andrunningheattolerancetest(RHTT)(y-axis)formales

(closedmarkers)andfemales(openmarkers).Dottedlinerepresentslineofequality.N=16(8M,

8F).

Figure3.4Peakrectaltemperature(Tre)andpeakheartrate(HR)duringtheIsraeliDefenceForce

heat tolerance test (IDF HTT) (x-axis) and running heat tolerance test (RHTT) (y-axis) formales

(closedcircles)andfemales(opencircles).Dottedlinerepresentslineofequality.N=16(8M,8F).

Figure4.1 Peak rectal temperature (Tre),peakheart rate (HR), sweat rate (SWR),andpeak skin

temperature(Tsk)duringRHTT1(x-axis)andRHTT2(y-axis),formales(closedmarkers)andfemales

(openmarkers).Dottedlinerepresentslineofequality.N=16(8M,8F).

Figure4.2Bland-Altmanplotswithmeanbias(solidline)and95%limitsofagreement(dottedline)

for peak rectal temperature (Tre), peak heart rate (HR), sweat rate (SWR), and peak skin

temperature(Tsk)formales(closedmarkers)andfemales(openmarkers).N=16(8M,8F).

Figure5.1Individualdataforvaluesofpeakrectaltemperature(Tre),peakskintemperature(Tsk),

peakheatrate(HR)andsweatrateSWRduringRHTT1(xaxis)andRHTT2(yaxis)formales(closed

markers)andfemales(openmarkers).Dottedlinerepresentslineofequality.N=25(12M,13F).

Figure6.1Rectal temperature (Tre)at5-min intervalsduring therunningheat tolerancetest for

males(left)andfemales(right).GreymarkersrepresenttheRHTT1,blackmarkersRHTT2andwhite

markerRHTT3.Mean±SD.N=16(8M,8F).

Figure6.2Heartrate(HR)at5-minintervalsduringtherunningheattolerancetestformales(left)

andfemales (right).Greymarkersrepresent theRHTT1,blackmarkersRHTT2andwhitemarker

RHTT3.Mean±SD.N=16(8M,8F).

Figure7.1Restingrectaltemperature(Tre)(left)andrestingheartrate(HR)(right)onD1,D5and

D10ofheatacclimation.Dottedlinerepresentslineofequality.N=10(5M,5F).

Figure7.2Hsp72mRNApreandpostsessiononD1,D5andD10ofheatacclimationinmalesand

females.Mean±SD.N=10(5M,5F).

XIV

Figure7.3Hsp72mRNApretopostsessiononD1,D5andD10relativetoD1preofheatacclimation

inmales(opencircles)andfemales(closedcircles).N=10(5M,5F).

Figure8.1Schematicoftheexperimentaldesign.

Figure8.2Plasmaconcentrationsof17ß-estradiolandprogesteronepriortoRHTT1andRHTT2in

theHACH intervention(whitebars)andRHTT1andRHTT2intheHAsauna intervention(blackbars)

andD22ofthemenstrualcycle(greybar).Mean±SD.N=9.

Figure8.3Rectal temperature (Tre)at5-min intervalsduringtheRHTTfor theHACH intervention

(left)andtheHAsaunaintervention(right).OpenmarkersrepresentRHTT1andtheclosedmarkers

representRHTT2.Mean±SD.N=9.

Figure8.4Heartrate(HR)at5-minintervalsduringtheRHTTfortheHACHintervention(left)and

theHAsaunaintervention(right).OpenmarkersrepresentRHTT1andtheclosedmarkersrepresent

RHTT2.Mean±SD.N=9.

Figure8.5Sweatrate(SWR)(left),sodiumchloride(NaCl)sweatconcentration(middle),andTreat

onsetofsweating(right)duringRHTT1(xaxis)andRHTT2(yaxis)intheHACHintervention(closed

markers)andHAsaunaintervention(openmarkers).Dottedlinerepresentslineofequality.N=9.

Figure9.1Correlationcoefficientsforassociationbetweenchangesinrectaltemperatureandbody

surfacearea(BSA),sumofskinfolds,relativeVbO2peak,relativeexerciseintensity,absoluteheat

production(Hprod,W),heatproductionrelativetoBSA(Hprod,W.m2),andheatproductionrelative

toBM(Hprod,W.kg-1).

Figure9.2Scatterplotillustratingtherelationshipbetweenthechangeinrectaltemperature(∆Tre)

andabsoluteheatproduction(Hprod,W)duringtheRHTT.N=30.

XV

Listofequations

Equation2.1Calculationforwetbulbglobetemperature(WBGT)(YaglouandMinard,1957)

Equation2.2Heatbalanceequation

Equation2.3Calculationformetabolicenergyexpenditure

Equation2.4Calculationformeanskintemperature(Tsk)

Equation3.1Calculationofbodysurfacearea(BSA)

Equation3.2Calculationofphysiologicalstrainindex(PSI)

Equation3.3Calculationofwholebodysweatrate(SWR)

Equation3.4Calculationofwholebodysweatraterelativetobodysurfacearea(SWRBSA)

Equation3.5Calculationofworkduringcycleergometry

Equation3.6Calculationofpowerfromworkduringcycleergometry

Equation3.7CalculationofrateofTreincrease

Equation3.8Calculationoftypicalerrorofthemeasure(TEM)

Equation3.9Calculationoftechnicalerrorasacoefficientofvariation(TE(CV%))

Equation3.10Calculationofintraclasscorrelation(ICC)

Equation3.11CalculationofCohen’sd(d)

Equation8.1CalculationforchangeinPlasmaVolume(∆PV)

XVI

Acknowledgments

Iwouldliketotakethisopportunitytothankthosewhohavemadethisthesispossibleandtothe

followingindividualsfortheirunconditionalsupportandguidancethroughthevariousstagesofmy

thesis.

Firstly,IwouldliketothankDrNeilMaxwellandProfJoDoustfortheirsupervisionandguidance

inundertakingmyDoctorateofPhilosophyinatopicthatIampassionateabout.Youhaveboth

actedasmentorsformeandsupportedmygrowthasanEnvironmentalPhysiologist.Youradvice

onbothresearchaswellasonmycareerhavebeeninvaluable.

ThankyoufellowTechnicalInstructor’sBen,James,Oli,andRosie,andPhDstudentsAsh,Carl,and

Cat for providing stimulating scientific discussions over extended coffee and lunch breaks.My

gratitudeisalsoextendedtothetechnicalteamattheUniversityofBrightonandLeeandJames

fromBedfordshireUniversityfortheirsupportwiththeHsp72mRNAanalysis.Iwouldalsoliketo

giveaspecialthankstoalloftheparticipantswhovolunteeredtheirtime,energyandcommitment

totheresearchprojects.

Aspecialthankstomyfamily.BothmymumHeatheranddadDavidhavealwayssupportedme

withmydecisiontoundertakeaPhD.Theyprovidedmewithagreatfoundationtolife,teaching

metobecaring,hardworking,dedicatedandindependent.MysisterLizzyforalwaysbeingthere

andofcourseDillanwhoseloveiscompletelyunconditional.

Lastbutnotleast,ImustacknowledgeBenedictforalwayskeepingmegroundedandhelpingme

torealisetheimportantthingsinlife.Benedicthasbeencentraltothecompletionofthisthesisas

hehasgivenmeconfidenceandmotivationwhentheywerelost.

XVII

Declaration

Ideclarethattheresearchcontainedinthisthesis,unlessotherwiseindicatedwithinthetext, is

originalwork of the author. The thesis has not been previously submitted to this or any other

universityforadegree,anddoesnotincorporateanymaterialalreadysubmittedforadegree.

Signed:

JessicaAnneMee

Date:18thMarch2016

XVIII

The following academic publications and conference proceedings have been due to the work

containedwithinthisthesis:

Academicpublications

Mee,J.A.Doust,J.andMaxwell,N.S.(2015).Repeatabilityofarunningheattolerancetest.Journal

ofThermalBiology49-50,91-97.

Mee,J.A.Gibson,O.R.Doust,J.D.Maxwell,N.S.(2015).Sexdifferencesinadaptationtoshort-and

long-termheatacclimation.ScandinavianJournalofMedicineandScienceinSports,25(Suppl.1),

250-258.

Mee,J.A.Peter,S.Doust,J.Maxwell,N.S.(2015).Restrictedsweatevaporationprecedingshort-

termheatacclimationacceleratesadaptationinfemales.ExtremePhysiologyandMedicine4(1),

A112.[Abstractonly].

Conferenceproceedings

Mee,J.A.Peter,S.Doust,J.Maxwell,N.S.(2015).Restrictedsweatevaporationprecedingshort-

term heat acclimation accelerates adaptation in females. International Conference of

EnvironmentalErgonomics(ICEE),Portsmouth,UK,28thJune-3rdJuly2015.

Mee,J.A.Gibson,O.R.Taylor,L.Tuttle,J.A.Watt,P.W.Doust,J.Maxwell,N.S.(2015)Sexcomparison

ofleukocyteHsp72mRNAupregulationduringheatacclimation.EuropeanCollegeofSportScience

(ECSS)Malmo,Sweden,24th–27thJune2015.

Mee,J.A.Gibson,O.R.Doust,J.Maxwell,N.S.(2014)Sexdifferencesinadaptationtoshort-and

long-term isothermicheatacclimation.Trainingandcompeting in theheatconference,Aspetar,

Doha,Qatar,23rd-24thMarch2014.

Mee,J.A.Doust,J.Maxwell,N.S.(2013).Reliabilityandvalidityofashortdurationrunningheat

tolerancetest.BritishAssociationofSportandExerciseScience(BASES)studentconference,Cardiff,

UK.

1

1. Introduction

_______________________________________________________________________________

Moderate ambient temperatures are known to present sufficient heat stress that result in an

elevated core temperature (Tc) and reduce endurance capacity (Galloway andMaughan 1997).

Duringexercise inahotenvironment,activemusclesperformworkcausingan increase inbody

heat content. These changes represent the rate of change in body heat storage,which in turn

reflectsthebalancebetweenmetabolicheatproduction,heatabsorbedfromtheenvironmentand

totalbodyheat loss(JayandKenny,2007).Exertionalheatstrokeischaracterisedbyarise inTc

beyond40°Ccoupledwithcentralnervoussystemdysfunctionandmultipleorgansystemfailure

(Casaetal.,2012).

TheworkbyNielsen(1996)providesdatatosuggestanenduranceathletemayexperienceuptoa

1°CriseinTcevery9-minwhenracinginhighambientconditions.ThisrateofriseinTcwouldresult

inarunnerreachingaTcof40°Cwithin25-30min,withtheimmediatedangersofheatexhaustion.

Dataarelimitedontheincidenceofexertionalheatillnessinlongdistancerunning,particularlythe

reportingofnon-fatalcases.The11.5kmFalmouthRoadRaceintheUSA,typicallyruninwarm

humidconditions(23.3°C,70%RH),reportsonaverage10–20casesper10,000entrantsperyr

(DeMartinietal.,2014).Incontrast,theTwinCitiesMarathoninUSA,runincoolerconditions(16°C,

60%RH)reportsanaverageof1caseofexertionalheatillnessper10,000finishers(Roberts,2000).

WhilehighambienttemperatureandRHareimportantfactorscontributingtowardsexertionalheat

illness, there are also incidences in cooler environments (Hawes et al., 2010; Roberts, 2006;

RobertsonandWalter,2010).TheGreatNorthRunintheUKin2009(18°C)reported55casesof

exertionalheatillnessamongthe55,000participants(Hawesetal.,2010),anincidenceratesimilar

tothatobservedattheFalmouthRoadRacewhereconditionsaremuchwarmer.

Martin (1997) reportedahigher incidencerate in females (36 incidence;3.63%)comparedwith

males(26incidence;2.18%);withfemalesaccountingfor58%ofthetotalnumberofcases(Martin,

1997).Questionably,oneofthemostmemorablemarathonfinisheswasofa39-yrs.old,female

Swissathlete,GabrielaAnderson-Schiess.ItwasapparentGabrielawassufferingwithaseriouscase

ofheatexhaustionduringthelatterstagesofthe1984Olympicinauguralfemales’marathon.She

limpedaroundthefinal400min5-minand44-s,refusingmedicalsupport,eventuallycrossingthe

finish line in2-hr48-minand45-s. The severityof this case is still apparent today,with similar

incidencessuchasthatof34-yr.old,female,Namibianathlete,BeataNaigambo.Duringthe2014

CommonwealthGamesmarathon,Beata staggereddown thehomestraightand faintedmeters

2

fromthefinishline,eventuallycollapsingoverthefinishlineunassistedin2-hr39-min23-s.This

body of evidence emphasises the severity of heat related illnesses in endurance athletes,

specificallyfemales.Further,ithighlightstheneedtodevelopeffectivescreeningproceduresand

heat-alleviating strategies, to support endurance athletes when training and competing in

thermally-challengingenvironments.

Heatintolerancemaybepermanentoracquiredandreferstoasyndromedescribingindividuals

whoareunabletoadaptproperlytoexercisinginahotenvironment.Heattolerancetests(HTT)are

currentlyusedtoevaluatemilitarypersonalthathavehadapreviousexperienceofexertionalheat

illness, to determine whether they are able to return to duty. The Israeli Defence Force (IDF)

developedaHTTwhichinvolves120-minwalkingonatreadmillatapaceof5km.hr-1anda2%

gradientinambientconditionsof40°Cand40%relativehumidity(RH).Inspiteofthesignificant

rolefortheIDFHTTtoscreenmilitarypersonnelforheatintolerance,theapplicationoftheIDFHTT

hasbeenquestionedinreferencetoenduranceathletesduetodifferencesinexerciseintensityand

duration,theclothing,andthecarryingload(Johnsonetal.,2013).Enduranceathletestypically

race forashorterdurationandatahigherexercise intensitycomparedwithmilitarypersonnel,

whomostcommonlyperformlowintensitymarchingforprolongedperiodsoftime.Therefore,the

rateofheatproduction,requirementforevaporativecooling,andchangesinTcdiffersubstantially

betweenthesepopulations.Theincidenceofexertionalheatillnessinenduranceathletes(Martin,

1997) raises the importance of developing a heat tolerance screening procedure that informs

medical personal about an endurance athlete’s current heat tolerance state, and is capable of

monitoringchangesinheattolerance.

Repeated heat exposure to a stressful thermal environment initiates a phenotypic adaptation

known as heat acclimation (HA), an element of which has been identified as thermotolerance

(Moseley,1997).HAimprovesthermalcomfort,submaximalexerciseperformance,andincreases

maximal aerobic capacity in the heat (Lorenzo et al., 2010). The benefits arise from enhanced

sudomotorandskinbloodflowresponses,plasmavolume(PV)expansion,cardiovascularstability

and an improved fluid balance (Poirier et al., 2015). Thermotolerance or acquired cellular

thermotolerance describes the cellular adaptation accompanying systemic changes induced by

successful HA (Horowitz, 2014; Magalhães et al., 2010). As a result, HA is the consensus

recommendation strategy to attenuate the physiological strain associated with training and

competingintheheat(Racinaisetal.,2015).However,HAitisnotroutinelyadoptedbyendurance

athletes; UK athletics currently only offer altitude training camps as part of their world class

performanceprogramme.

3

Controlled hyperthermiaHA ensures equal thermal strain is placedon participants during each

session,asitinvolveselevatingandmaintainingasteadystateTcabovethesweatingthreshold(Fox

et al., 1963a). This method has become increasingly popular since it offers more complete

adaptationdue to theprogressiveoverloadapproach comparedwith traditional, fixed-intensity

protocolswhichuseaconstantoverloadapproach(Gibsonetal.,2015b).However,thereremains

apaucityofdataconcerningbestpractice forHA in females; consequently, femaleathletesare

attendingwarmweather trainingcampsor implementingHAstrategiesbasedondatacollected

frommale participants. The implications of this are that femalesmay be adopting sub-optimal

strategiesthatprovidelittlebenefittoalleviatingphysiologicalstrain.

Sex has traditionally been considered an independent modulator of temperature regulation

(Kaciuba-UscilkoandGrucza,2001;Kenney,1985;Nunneley,1978),howevertheseobservations

may be confounded by inherent physical differences between the sexes. More recently, sex

differences in temperature regulation during exercise have been reported, irrespective of

confounding differences inmetabolic heat production andphysical characteristics (Gagnon and

Kenny, 2012, 2011). Interestingly, these differences only become evident above a certain

requirement for heat loss and are solely attributed to peripherally mediated control of the

sudomotor function. Furthermore, fluctuations in hormonal concentrations associatedwith the

menstrualcyclehaveaknowneffectonthermoregulationinfemales.Duringthelutealphaseof

themenstrual cycle there is anelevation inprogesterone concentrationwhichmodifies central

regulatorymechanisms resulting in alterations in basal Tc, the body temperature threshold for

sweating,andthebodytemperaturethresholdforcutaneousvasodilationontheforearm,chest,

back,forearm,andthigh(Inoueetal.,2005).Thesesexdifferencesinthermoregulatoryresponses

toacuteexerciseheatstress,raisesthequestionofwhetherfemalesadapttochronicheatexposure

differently tomales. Thus, research investigating females’ phenotypic and cellular responses to

controlledhyperthermiaHAovershort-andlong-termtimescalesisrequired.

Recently,novelHAstrategieshavebeeninvestigatedinmaleparticipants.Thesemethodsinvolve

permissivedehydration(Garrettetal.,2012;Nealetal.,2015),saunabathing(Scoonetal.,2007;

Stanley et al., 2015), and hotwater immersion (Zurawlew et al., 2015). Thesemethods aim to

establish an accelerated adaptation via targeting adaptation pathways including, PV expansion,

increased sudomotor function, and altered cutaneous vascular function. Once the temporal

patterningtocontrolledhyperthermiainfemaleshasbeendetermined,furtherresearchisrequired

toestablishnovelHAstrategiesthataccelerateadaptionresponsesinfemales.

4

Thisintroductionhasoutlinedtheincidenceofheatrelatedillnessinrunners,particularlyfemales,

the requirement to develop a running HTT (RHTT), the clear need for research into females

phenotypic and cellular adaptations to controlledhyperthermiaHA, and introduced the ideaof

investigating novel HA strategies to accelerate adaptation in females. This thesis is therefore

presentedinthefollowingchapters.

Chapter2reviewstheliterature,discussingacuteandchronicresponsestoheatstress.Thefirst

partoftheliteraturereviewconsidersthephysiologicalandthermoregulatoryresponsetoanacute

heat exposure, discussing individual differences, with a particular focus on any potential sex

differences.Thesecondpartoftheliteraturereviewfocusesonheatillnessesandtheincidence

rateinrunning.ThethirdpartoftheliteraturereviewdiscussesHAprotocolsandthetimecourse

ofadaption.ThefourthsectionfocusesonthepotentialsexdifferencesinresponsestoHA.The

final sectionof this chapteroutlines the thesis aims, researchquestions andhypothesesof the

experimentalstudies.ThesearchwasrestrictedtofulltextarticlespublishedinEnglishlanguage

peer reviewed journals. Where possible information was limited to articles using human

participants,howeverdue to thenoveltyof someof researchpresented, animaldatahasbeen

used.

Chapter3describesthecommonmethodsusedacrossmultipleexperimentalchapters.

Chapter4presentsthefirstexperimentalchapter(Study1)wherebytherepeatabilityofaRHTT

wasassessed.Thefindingsfromthisstudyinformsfutureexperimentalchaptersbyquantifyingthe

measurement error associated with the RHTT to support accurate conclusions regarding the

magnitudeofadaptationobserved.

Chapter5presentsthesecondexperimentalchapter(Study2)wherebythesensitivityoftheRHTT

toquantifychangesinheattolerancefollowingshort-termHA(STHA)wasassessed.

Chapter6usestheRHTTtoquantifythechangesinphysiologicalandthermoregulatoryresponses

followingcontrolledhyperthermiaHAovershort-andlong-termtimescalesinmalesandfemales

(Study3). This chapterprovidesnovelHAdataon females anddescribes thedifferences in the

temporalpatterningbetweenmalesandfemales.

Chapter7presentsthefourthexperimentalchapter (Study4)describingtheheatshockprotein

generesponses(Hsp72mRNA)tocontrolledhyperthermiaHAovershort-andlong-termtimescales

inmalesandfemales.

5

Chapter 8 describes the findings from the final experimental chapter (Study 5). Following the

identificationthatfemalesrequiredlong-termHA(LTHA)toestablishareductionincardiovascular

andthermoregulatoryresponsesinchapter6(Study3),anovelmethodofcombiningasauna-like

exposure with controlled hyperthermia HA was investigated to establish its effectiveness in

acceleratingadaptationresponsesinfemalesoverashort-termtimescale.

Chapter9discussestheindividualandcumulativefindingsofalltheexperimentalchapterstoform

thegeneraldiscussion.Thefirstpartofthegeneraldiscussionwillreviewtheprinciplefindingsfrom

the experimental chapters presented. The second part of the general discussionwill provide a

mechanisticoverview,focusingspecificallyonheattolerance,thetemporalpatterningofHA,and

acceleratingtheadaptioninfemales;involvingretrospectiveanalysesofpooleddatafromwithin

thethesis.Thischaptercloseswithdiscussingpotentialfuturedirectionsforresearchinthefield,

inadditiontoreviewingthepracticalapplicationofthepresenteddata.

6

2. Literaturereview

________________________________________________________________________________

2.1. Environmentalheatstressandexchange

Heatstress refers toboththeenvironmentalandmetabolicconditions that typically increaseTc

(Sawkaetal.,2011a).Heat stress is categorisedascompensableheat stress,oruncompensable

heatstress.CompensableheatstressreferstoasteadystateTc,wherebytheheatlossmatchesthe

heatproduction(Sawkaetal.,2011a).Uncompensableheatstressreferstoanimbalancebetween

theevaporativerequirementandtheevaporativecapacityofanindividual(Cheungetal.,2000).

Duringuncompensableheatstress,anindividualisunabletodissipatethemetabolicheat,causing

acontinualriseinTc,untileitherexhaustionoccurs,ortheseverityoftheenvironmentalconditions

decreases(Cheungetal.,2000).

To quantify environmental heat stress in occupational, military and sports applications a user-

friendlymeasureknownaswetbulbglobetemperature(WBGT)iscommonlyused(Budd,2008).

WBGTisanempiricalindexofclimaticheatstresswhichcanbeusedtoquantifytheheatstress

(Sawkaetal.,2011a).WBGTcanbecalculatedusingEquation2.1.

Equation2.1Calculationforwetbulbglobetemperature(WBGT)(YaglouandMinard,1957)

WBGT=0.7Tw+0.2Tg+0.1Td

Where:Tw=wetbulbtemperature,Tg=blackglobetemperature,Td=drybulbtemperature

TheWBGT has a number of limitations which questions its validity, accuracy and applicability.

Specifically,theWBGTdoesnotmakeanyconsiderationsforclothingormetabolicrate,asaresult,

it cannot predict heat exchange between a person and the climate (Budd, 2008). Despite its

limitations,theWBGTisthemostwidelyusedindex,sinceitiseasytouseandisareliablemeasure

ofheatstress(Budd,2008).ManygeographicalregionsexperienceWBGTabove29°Cduringthe

summermonths.Thisiscausedbyahighhumidityand/orahighairtemperatureandsolarload,

asreflectedbyahighwetbulbtemperatureandblackglobetemperature,respectively(Sawkaet

al.,2011a).Theuniversal thermalclimate index(Fialaetal.,2012)providesanalternativetothe

WBGT,byintegratingpredictiveformulasofheatstrainbasedonactivitiesconductedindifferent

7

temperaturesatvaryingconstantmetabolicheatproductions.Whiletheuniversalthermalclimate

indexmayultimatelyofferamorecomprehensiveandrobustevaluationoftheriskofphysiological

heatstrain,itismorecomplexandexpensivetoadopt.Toensurethatanyheatstressmonitoring

system is widely adopted across all settings, solutions must be relatively simple to use and

implement, while being affordable (Périard et al., 2016). Additional research is warranted to

developamoreappropriatetooltosupportgoverningbodiesinensuringthehealthandsafetyof

athletesinhotenvironmentalconditions.

Physicalexerciseincreasesmetabolicheatproductionabovetherestingrate(<100W).Depending

uponthetypeofexercise,approximately80to90%ofenergyproduced isreleasedasheatand

thendispersedaroundthebody,mostlythebloodstream(JayandKenny,2007).Itisnecessarythat

the heat is dissipated from the body to avoid heat storage, with a resultant increase in Tc.

Cardiovascularadjustmentsoccurinordertoredirectthebloodflowfromthebody’scoretothe

peripheryforsubsequentheatdissipation(Nyboetal.,2014).Theheatdeliveredtotheperiphery

is transferred to the environment by dry (radiative, conductive and convective) and wet

(evaporative)heattransferpathways.Theeffectivenessoftheheattransferpathwaysisdependent

upon the biophysical properties of the environment. This includes the temperature differences

betweentheskinsurfaceandtheenvironment,theevaporativepotentialandthesolarconditions

(Sawkaetal.,2011a).

2.1.1. Dryheattransferpathways

Thenon-evaporativeavenuesofheatexchangesarecollectivelycalleddryheatexchange;these

includeconduction,convectionandradiation(figure2.1).Conductiveheattransferoccurswhen

twosolidobjectsareindirectcontactwitheachotherandthereisatemperaturegradientbetween

thebody’ssurfaceandacontactingsolidobject.Whenperformingexerciseinastandingposition

such aswalking and running the conductive heat exchanges isminimal since the contact point

betweenthebody’ssurfaceandthegroundisverysmall(KennyandJay,2013).Convectionisthe

heat exchange between the surface of a solid object and a fluid, including air andwater.Heat

transfer via convection to air or fluid occurs when the air or fluid temperature is below the

temperatureofthebody.Anincreaseinwindorfluidcurrentfacilitatesagreaterrateofconvective

heattransfer;furthermore,theheatcapacityofwaterisfargreaterthanairincreasingconvective

heat transfer. Radiation is the heat exchange between a solid object and a large natural, or

manufacturedobjectsuchasthesunoraheater(Sawkaetal.,2011a).

8

2.1.1. Wetheattransferpathways

Evaporativeheat lossoccurswhen liquid changes intowater vapour. Theevaporativeheat loss

capacityisdeterminedprimarilybythewatervapourpressuregradientbetweenthebody’ssurface

andtheenvironment.Thiscanbemodifiedbytheenvironmentandclothing(Sawkaetal.,2011a),

inadditiontophysiologicalalterationinsweatglandactivityandoutput(Taylor,2014).Evaporative

coolingistheprimarymeansofheatremovalduringexercise.Evaporativecoolingistheonlymeans

forheatremovalduringexerciseinanambienttemperaturethatisequalto,orgreaterthanskin

temperature(Tsk).

Figure2.1Avenuesforheatexchangewhenperformingexercise.AdaptedfromGisolfiandWenger,

(1984)

2.1.2. Heatbalance

Theinteractionbetweenfactorsthatincreaseordecreaseheatproductionandheatdissipationand

theresultantimpactuponbodyheatstorageisevidentintheheatbalanceequation(Equation2.2).

Metabolicheatproductionistypically<100Watrestand>1000Wduringhighintensityexercise.

Therateofbodyheatstoragerepresentsheatgainifpositiveandheatlossifnegative.

9

Equation2.2.Heatbalanceequation

S(watts)=M–(±W)±(R+C)±K–E

Where:S=rateofbodyheatstorageinwatts,M=rateofmetabolicenergyproduction,W=mechanical

work,whetherconcentric(positive)oreccentric(negative)exercise,R+C=rateofradiantandconvective

energyexchanges,K=rateofconduction,E=rateofevaporativeloss.

Metabolicheatproductionisthedifferencebetweenmetabolicrateandthefractionofthisenergy

thatisusedtocreateexternalwork.Thehumanbodyisinefficientatperformingexternalworkand

thus80%-100%oftheenergyproducedisreleasedasheatenergyinsidethebody.Forexample,if

aparticipantiscyclingat100W,400Wofthe500Woftotalenergyproductionwillbeheatenergy

insidethebody.

Indirectcalorimetry

Indirectcalorimetry is the traditionalmethod forestimatingmetabolicenergyexpenditure.This

approachrequiresthemeasurementoftherateofoxygen(O2)consumptionandcarbondioxide

(CO2)production.Usingtheenergyequivalentforthefulloxidationofcarbohydrates(21.13kJper

LofO2consumed)andfats(19.63kJperLofO2consumed),metabolicenergyexpenditurecanbe

subsequentlycalculatedusingEquation2.3(KennyandJay,2013).

Equation2.3Calculationformetabolicenergyexpenditure

M=[VbO2((((RER-0.7)/0.3)ec)+(((1.0-RER)/0.3)ef)]/60

Where:VbO2ismeasureinL.min-1;ecisthecaloricequivalentperlitreofoxygenfortheoxidationof

carbohydrates(21130J)andefisthecaloricequivalentperlitreofoxygenfortheoxidationoffat

(19630J).

When direct calorimetry is unavailable, indirect calorimetry is the most precise method of

estimating rate of metabolic energy expenditure and subsequently metabolic heat production

(KennyandJay,2013).However,despiteheatproductionincreasingalmostimmediatelyafterthe

startofexercise,O2consumptionincreasesexponentiallywithatimeconstantof15-30s.Assuch

>1-minofexerciseatthesameexternalworkloadisrequiredtoattainanelevatedsteadystateof

10

O2consumption(AstrandandSaltin,1961;Cheuvrontetal.,2010;WhippandWasserman,1972).

Thus,thedifferencebetweentheactualrateofmetabolicheatproductionatthetissueleveland

itsmeasurementfromanalysingexpiredairshouldbeacknowledged.

Directcalorimetry

Duringboutsofheat imbalance, suchasduring theonsetofexercise,a summationof theheat

exchangeviaconvection,conduction,radiationandevaporationmustbeobtainedandcompared

toconcurrentmeasurementsofmetabolicenergyexpendituretodeterminethequantityofheat

storedinsidethebody(KennyandJay,2013).Themostaccuratemethodforquantifyingtherate

of net heat loss is with direct whole body calorimetry (Jay and Kenny, 2007). Whole body

calorimetry is the simultaneousmeasurementsof the internalheatgeneratedby thebodyasa

resultofanaerobicandoraerobicmetabolismandthetotalheatlosttotheenvironment(Jayand

Kenny,2007).Thus,ifanimbalanceoccursbetweenmetabolicheatproductionandnetheatloss

to the environment, the time dependent differences between thesemeasurements provide an

estimateofthechangeinbodyheatcontent.

2.2. Heatstrain

Heatstrainreferstoanyphysiologicalconsequencesofheatstress(Sawkaetal.,2011b).Measuring

Tc is fundamentaltotheexperimentalstudyoftemperatureregulation inhumans(Sawkaetal.,

1993).Tc istypicallycontrolledat37.0±0.5°Cduringrest.However,Tcvariesbetweendifferent

bodyregionsdependingonthemetabolicrateofthelocaltissues,thesourceandthevolumeof

localbloodflow,inadditiontothetemperaturegradientsbetweenconnectingbodyregions(Sawka

etal.,2011a).

Inarestedstate,theinternalorgansandvisceraproduceapproximately70%ofmetabolicheat,

howeverduringmovement,theskeletalmusclesproduceapproximately80-95%ofmetabolicheat

(Sawkaetal.,2011a).Metabolicheat sourcesarealtered fromrest toexercise.Asa result, the

calculatedchangeinbodytemperaturemaybedisproportionateinonebodyregioncomparedwith

another.Forexample,whenhumansareinarestedstatetheirTc(37.0±0.5°C)istypicallyhigher

than muscle temperature (36.0 ± 0.5°C), however during exercise, the temperature of active

skeletalmuscleusuallyexceedsthatofTc(Kennyetal.,2008)(figure2.2).

11

Figure2.2Meanchangesincoretemperature(A)andmuscletemperature(B)duringandfollowing

60-minexercise.N=8(6M,2F)TakenfromKennyetal.,(2008).

2.2.1. Measurementofcoretemperature

ThemeasurementofTcvariesdependingontheinternalmeasurementsite(Lefrantetal.,2003).It

isessentialthatthemethodadoptedtomonitorTcisconvenientandunbiasedbyenvironmental

conditions (Sawka et al., 2011a). Furthermore, there is a requirement that the measurement

techniquemirrors small changes in arterial blood temperaturequickly, toaccurately reflect the

temperatureofbloodperfusingthehypothalamus(Lefrantetal.,2003).Themostcommonlyused

measurementsites include, therectum,oesophagus,andgastrointestinal tract,butalso include

auditory-canal. Figure 2.3 presents oesophageal, rectal, and intestinal temperature measured

simultaneouslyduringrest,submaximalsupinecyclingexerciseat40%O2uptake(VbO2)peakand

65%VbO2peak,andduringpassiverecovery.

12

Thelocationofrectaltemperature(Tre)isconsideredthemostpracticalandaccurateformeasuring

Tc (Moran andMendal, 2002). Furthermore, Tre is typically very easy to administer and causes

minimaldiscomforttotheparticipants.Heatexchange intherectumisduetotheneighbouring

tissues,ratherthantheregularperfusionofhotblood;thethermalinertiaandpoorheatremoval

results in a stable measurement. However, Tre responds slower to the rapid changes in Tc

experiencedduringtheonsetofexerciseandhasameandifferenceof0.4±1.0°Ccomparedwith

thepulmonaryartery(Robinsonetal.,1998).Oesophagealtemperatureisoftenconsideredtobe

the best non-invasive method to determine Tc because of its deep location, close to the left

ventricle, the aorta, and to the blood flow to the hypothalamus. Furthermore, oesophageal

temperaturerespondsrapidlyandquantitativelytochangesincentralbloodtemperature(Lefrant

etal.,2003)andthepulmonaryarterytemperature(0.0±0.5°C)(Robinsonetal.,1998).However,

oesophageal probes are sometimes deemed as undesirable in many settings because of the

difficultly in inserting the thermistor, irritation to the nasal passage and general participant

discomfort(MoranandMendal,2002).Thegastrointestinalpillprovidescomparablesteadystate

TcmeasurementstoTre.However,thegastrointestinalpillrespondsmoreslowlytothechangesin

Tc than oesophageal temperature (Lee et al., 2000). Furthermore, the gastrointestinal pill

temperaturevalueshavebeenshowntobemorevariableovertimesincethepillistravellingwithin

thegastrointestinaltract(ByrneandLim,2007).

Auditory-canalisanotherestablishedmethodtotrackTc(Cooperetal.,1964;Edwardsetal.,1978;

Haywardetal.,1984)albeitwithanoffsetandaphasedelay(Tayloretal.,2014).Auditory-canal

hasbeenreportedtobecomparabletoTre(Ilsleyetal.,1983).However,surfacetemperaturesare

influencedbyambienttemperatureresultinginareducedaccuracyofauditory-canaltemperature

totrackTc(Lowetal.,2007)

Inconclusion,itappearsthatTre,duetoitsinherentstability,isthepreferredmethodtoaccurately

trackTcwithminimalmeasurementartefact,practicality,andminimalparticipantdiscomfort.

13

Figure2.3Oesophageal,rectal,andintestinaltemperaturemeasuredsimultaneouslyduringrest,

submaximal supine cycling exercise at 40% VbO2 peak and 65% VbO2 peak, and during passive

recovery.N=7(5M,2F).

Notes:Valuesaremean±standarderror.Takenfrom(ByrneandLim,2007;Leeetal.,2000).

2.2.2. Measurementofskintemperature

ThemeasurementofTskisconductedforseveralreasons.Firstly,themeasurementofTskenables

thecalculationofmeanbodytemperaturetopermitthedeterminationofheatstorage(Jayand

Kenny, 2007). Tsk measurements can also be used to estimate radiative and convective heat

exchange,skinconductance,andskinbloodflowrequirements.Furthermore,Tskmeasurements

can be used to inform the Tsk input to the thermoregulatory controller (Sawka et al., 2011a).

AlthoughTskcanbeassessedeasily,itcanalsobeproblematic,sincechangesinTskcanbecaused

byphysiologicaladjustmentssuchasalterationsinthecutaneousbloodflow,secretionofsweat

andevaporation,orchangestotheenvironmentalconditions(Sawkaetal.,2011a).Ensuringthat

the thermistors stay in thermal contact with the skin is paramount to the accuracy of Tsk

measurement(Smithetal.,2010).Thermistorshavebeenreportedtobearobustandaccurateto

0.05°Cacrossarangeofwaterbathtemperature(10-40°C)(Harper-Smithetal.,2010)butmore

variablewhenexercising(technicalerrorofmeasure[TEM)]=0.3°C)(Jamesetal.,2014).

14

MeanTskisoftencalculatedbasedonmultiplemeasurementstobetterreflectthechangesinTsk.

MeanTsk represents the sumof theweighted individualmeasurementsofTsk. Theweightingof

meanTskisestablishedbasedonthepercentageofbodysurfacearea(BSA)thatisrepresentedby

themeasurementsite(MitchellandWyndham,1969).TwelvetofifteenTskmeasurementsiteshave

beenusedtocalculatemeanTsk.However,investigatorsreducedthenumberofTskmeasurement

sitesrequiredtoobtainavalidestimateofmeanTsk.Whenitisproblematictomeasureaccurately

a large number of sites, the equation (Equation 2.4) introduced by Ramanathan should be

considered(Ramanathan,1964).

Equation2.4.Calculationformeanskintemperature(Tsk)

Tsk=0.3·(Tchest+Tarm)+0.2·(Tupperleg+Tlowerleg)

Where:Tchest,themidpointofthepectoralismajor;Tarm,themidpointofthetricepsbrachiilateral

head;Tupperleg,rectusfemoris;Tlowerleg,gastrocnemiuslateralhead.

In contrast, mean Tsk can be calculated based on the regional weighting of the skins thermal

sensitivity. The thermal receptors within the skin are not evenly distributed. As a result, the

warmingofbodyregionswhichhavethemostthermalsensors,ultimatelyhasthelargestinfluence

ontheeffectorresponses(Sawkaetal.,2011a).Thus,itissuggestedthatcalculatingmeanTskusing

thermal sensitivityweightingsmayprovidea superior index,whichmoreaccurately reflects the

peripheral input to the thermoregulatory controller. However, there remains some level of

disagreement as towhether themean Tsk values actually differ if calculated based on regional

thermalsensitivityweightingsorregionalBSAweightings(Libertetal.,1984).

2.2.3. Bodyheatcontent

Intheeventofanimbalancebetweenheatproductionandheatlossthereisarateofchangein

body heat storage and subsequently, a change in body heat content. Body heat content is the

productofmeanbody temperatureand theaverageheat capacityof thebody tissues (Jayand

Kenny, 2007). The determination of body heat content is of fundamental importance when

assessing the exposure of the human body to a hot environment thatmay result in a thermal

imbalance.Themeasurementofbodyheatexchangeusingsimultaneousmeasuresoftotalheat

productionandheatloss,intheoryistheonlymethodwherebybodyheatcontentcanbedirectly

determined(JayandKenny,2007).Therefore,bydefinitionthedifferencebetweenmetabolicheat

15

productionusingindirectcalorimetryandthetotalheatlostfromthebodyusingdirectcalorimetry

is theonlymethodtocalculate the rateofchange inbodyheatstorageandbodyheatcontent

accurately(JayandKenny,2007).Changesinbodyheatcontentarerarelymeasureddirectlyusing

calorimetry since this approach requires very sophisticated and expensive equipment and

instrumentation(JayandKenny,2007).Typically,changesinbodyheatcontentareestimatedusing

thetwocompartmentthermometrymodelwhichinvolvesthemeasurementofchangesinmean

bodytemperatureestimatedfromweightedTcandmeanTskmeasurements(JayandKenny,2007)

(figure2.4).ForthismethodtheTcandmeanTskvaluesareweightedbytheirrelativesizewhich

variesreciprocallywithcutaneousvasodilationandvasoconstrictionwithcore/skinweightingsof

0.90/0.10,0.79/0.21,and0.66/0.34inhot,temperate,andcoolconditions(Sawkaetal.,2011a).

The three-compartment thermometry model (figure 2.4) is reported to be a superior model

comparedwiththetraditional two-compartmentmodelduringexercise intemperateandwarm

conditions(JayandKenny,2007).Thethree-compartmentthermometrymodelusesmeasuresof

Tc, Tsk, and muscle temperature collectively, to predict changes in mean body temperature.

However, even when including invasive muscle temperature measurements, the three-

compartmentthermometrymodelonlyaccountsforapproximately50%ofthevarianceofmean

bodytemperature(JayandKenny,2007).Thisevidencesuggeststhattemperaturemeasurements

of more tissues intermediate to the core and shell, are required to provide a more accurate

thermometricderivationofbodyheatcontent.Subsequently,atwo-compartmentalthermometry

modelwasintroduced(JayandKenny,2007).Thismodelonlyaccountsforapproximately56%of

thevarianceofmeanbodytemperaturefromcalorimetrymeasurements.Thesefindings,suggest

that thermometry provides inaccurate estimates of mean body temperature changes, and

therefore thermoregulatory models to determine body heat content are likely flawed. Thus,

thermometrydeterminationofbodyheatcontentshouldbeusedwithcautionandonlyemployed,

whenusingarepeatedmeasuresdesign.

16

Figure2.4The2-compartmentthermometrymodelandthe3-compartmentthermometrymodelforchangeinmeanbodytemperatureandchangeinbodyheatcontent.

Adaptedfrom(JayandKenny,2007;KennyandJay,2013).

17

2.3. Behaviouraltemperatureregulation

Humans typically regulate their Tc within a narrow range between 35°C and 41°C using a

combination of behavioural and physiological responses. Thermoregulatory behaviour, is the

regulationofbodytemperaturebyacomplexpatternofmuscularskeletalresponsestoheatand

cold,whichmodifytheratesofheatproductionand/orheatloss(BlighandJohnson,1973).

Behavioural temperature regulation is often the first line of defence in maintaining body

temperature and involves a number of conscious alterations in behaviour. Examples of such

behaviours include,butarenot limitedto,modifying theexercise intensity,addingor removing

clothing,alteringbodyposition,andseekingshadeorshelter(Schladeretal.,2011b;Tuckeretal.,

2006).Schladeretal.,(2011a)reportedashortertimetoexhaustion(20.3±3.4minversus23.2±

4.1min),higherpeakTre(39.4±0.3°Cversus39.1±0.4°C),andhigherratingsofperceivedexertion

(18versus16)whenexercisewasperformedatafixedintensitycomparedwithself-pacedexercise,

in an uncompensable environment. Thus, when self-paced exercise is performed in an

uncompensableenvironment,itislessphysiologicallydemandingsincethevoluntaryreductionsin

exerciseintensityreducemetabolicheatproduction,improvethermalcompensability,andincrease

exerciseduration.Thisevidenceissupportedbythehighincidencerateofexerciseinducedheat

strokewhenhighmotivationandexternalfactorsdictateexerciseintensityandalowerincidence

whenpeopleexercisealoneorself-paced(Armstrongetal.,2007;Epsteinetal.,1999).Athletes

oftenignorebehavioursdesignedtosupportthermoregulationduetoahighlevelofmotivation

duringcompetition.

2.4. Physiologicaltemperatureregulation

Physiological temperature regulation is controlled by responses that are independent to our

conscious voluntary behaviours. Examples of such responses include, but are not limited to,

modifyingthebodyheatdistributionviaredistributingbloodflowthroughthecoreandtheskinby

cutaneousvasodilationandvasoconstriction,andalteringtherateofmetabolicheatproduction

through involuntary behaviours such as shivering, and sweating. There are several theories

developedwhichtrytoexplainhumanthermoregulatorycontrol;theseincludetheadjustableset

pointmodel,thereciprocalinhibitionmodel,andtheheatregulationmodel.

18

2.4.1. Adjustablesetpoint

Thetraditionalmodelofthermoregulatorycontrol,knownasthesetpointmodelwasintroduced

byHaroldHamel(figure2.5).Thissystemassumesthatafferentsignalsfromthecoreandperiphery

are combined into a thermal signal transmitted through the central nervous system and likely

withinthepreopticanteriorhypothalamus(Hammeletal.,1963).Thismodelisinpartinformedby

warmandcoldreceptorsunderthehumanskin.Thesereceptorsaretypicallylocatedatadepthof

0.3-0.6mmand0.15-0.17mm,respectively(Boulant,2006).Furthermore,neuralsupportforthis

model is highlighted by the observation that the hypothalamus holdswarm and cold sensitive

neuronswhichaltertheirfiringratebasedoneitherwarmingorcooling.Thisthermalsignalisthen

comparedtoaninternallystoredthermalsignal,resultingintheappropriateactivationofheatloss

orheatgaineffectorresponses(Hammeletal.,1963).

Acommonargumentincontradictionoftheadjustablesetpointmodelisthatitdoesnotexplain

any deviations from the reference temperature stored within the central nervous system. For

example, during the luteal phase of the menstrual cycle, baseline temperature is regulated

approximately 0.3-0.5°C higher than compared with the follicular phase (Coyne et al., 2000).

Similarly,duringafeverbodytemperatureisraisedandgenerally,appropriatelydefended.Theset

pointmodeloftemperatureregulationarguesthatthereisnorequirementforaconstantreference

temperature;instead,thesetpointtemperaturemayvaryoverabroadrangebasedontheactivity

ofwarmandcoldsensitiveneurons(Cabanac,2006).

19

Figure2.5Schematicdiagramofthethermoregulatorycontrolsystem.AdaptedfromSawkaetal.(2011).

20

2.4.2. Reciprocalinhibition

Thereciprocalinhibitionmodeloftemperatureregulationutilizesasimilarneuralstructuretothe

adjustablesetpointmodel,butexplainstemperatureregulationslightlydifferently.Thereciprocal

inhibitionmodelarguesacomplex integrationbetweensensoryreceptorsandthermaleffectors

forheatlossandheatgain(figure2.6)(Bligh,2006).Asopposedtoamoredirectintegrationofa

composite signal and comparison to a set signal, as suggestedby the adjusted setpointmodel

(Hammeletal.,1963).Thismodelassumesthatwarmandcoldsensorinputsareintegratedand

summed to produce a net heat loss or heat gain response, respectively. However, this model

assumesa“nullzone”oftemperatureregulationwhereheatlossandheatgaineffectorresponses

donotsufficiently respond, sincevasomotorchangesmaintain thermal stasis (Bligh,2006).This

model integrates temperature regulation into a large picture of whole body physiological

homeostasis by assuming the ability of a variety of non-thermal factors to affect temperature

control(Mekjavic,2006).

Figure2.6Thereciprocalinhibitionmodelofthermoregulation.Adaptedfrom(Bligh,2006).

Thereciprocalinhibitionmodeladdsanadditionallayerofcomplexitytotemperatureregulation.

Theactivityofsensorsinterconnectswiththepathwayoftheothersensors,toprovideaninhibitory

stimulus(Bligh,2006).Forexample,strongstimulationofwarmresponsiveneuronsactivatesthe

heatlosseffectorpathwaysandinhibitsthecoldsensitiveneuronsandheatgainpathways.The

reciprocalinhibitoryprocesspermitsthenetthermalsignalstoevokeaneffectorresponseunless

thesignalisnotstrongenoughwhereminorvasomotorchangesoccur(Bligh,2006).Similarly,non-

thermalstimuluscanproduceeitheranexcitatoryorinhibitoryeffectoneithersetofpathways,

helpingtoexplaintheshiftingofTcthresholdswithindividualchanges(Bligh,2006).Animplication

21

of thismodel, is that there isnocentral comparator signalas impliedby theadjustedsetpoint

modelwhichpermitseasiertheoreticalabilityforTcshifts(Sawkaetal.,2011a).Anatomicalsupport

for this ideacanbeseen inclinical tests inwhich thehypothalamus is impairedorblockedand

thermoregulatorycontrol,althoughcoarser,isstillpossible(Sawkaetal.,2011a).

2.4.3. Heatregulation

Theheatregulationmodelisdifferentinthetheoreticalconstructfromtheadjustedsetpointand

thereciprocalinhibitionmodelsofphysiologicaltemperatureregulation.Theunderlyingprinciple

oftheheatregulationmodelisthattheoverallheatstorage,orthehomeostaticnetbalanceofheat

gainedversusheat lost is theregulatedvariable.Suchthatbodytemperature isnotaregulated

variable, it is the resultof thebody’sattempt tomaintaina stableoverallheatbalance (Webb,

1995).AsopposedtosensingandintegratingperipheraltemperatureandTc,thebodyusesheat

flowandthetemperaturegradientacrosstheskinsurfaceasitsprimaryafferentinputs.Thecentral

integrationof theheatbalanceoperateswith feedback fromheat lossand feedforwardcontrol

fromheatproduction,drivingthephysiologicalresponsesthatdefendbodyheatcontent(Webb,

1995).

Theprimarystrengthoftheheatregulationmodeloftemperatureregulationisthatitissensitive

tothedeviationsinbodytemperature.Asapracticalexample,whenacyclistbeginsexercisingthere

isaconcurrent increase inTc,this is followedbyastabilisationofTcuntilexercise isterminated

(figure2.7).Heatproductionrespondsmorequicklycomparedtotheheatdissipationmechanism

however,eventuallytheheatlossmechanismsmatchestherateofheatproduction,resultingina

stable elevated Tc.Whenexercise is terminated, Tc remains elevated for a periodof timeeven

though heat dissipating responses are reduced rapidly (Kenny et al., 2008) (figure 2.7). This is

difficulttoaccommodateinthetemperatureregulationmodelssince itwouldassumethatheat

dissipating responseswould remainelevated throughout the recoveryperioduntilTc returns to

resting levels. The primary argument against the heat regulation model is the anatomical

requirement for heat flow sensors which are anatomically difficult to demonstrate, whilst

accountingforthepresenceoftemperaturesensitiveneuronsthroughouttheperipheriesandthe

central nervous system. In response supporters for the heat regulation model argue that the

thermal receptors throughout the body representmultiple thermal sensors, and the difference

betweenthesethermalsignalscanbecomeintegratedasratesofheatflow.

22

Figure2.7Meanwholebodycalorimetrydataforrateoftotalheatproductionandrateofnetheat

loss after60-min rest, 60-min cyclingat70Wand60-minof inactive recovery.N=8 (6M,2F).

AdaptedfromKennyetal.(2008).

These threemajormodels of thermoregulatory control have been advanced over the years to

develop our understanding of thermal homeostasis. The first two models discussed, suggest

temperatureistheregulatedvariableandsharesimilarideasconcerningtheunderstandingofthe

neuralarchitecture.However,thethirdmodelisfundamentallydifferentfromthefirsttwomodels

in that rather than temperature per se, body heat content is the regulated variable and body

temperature is aby-productof that regulation.All thesemodelshave their strengths,buteach

remains imperfect, thus more research is required to continue the developments in our

understandingofthermalhomeostasis.

2.5. Physiologicalresponsestoheatstress

Thereareseveralprinciplethermoregulatoryresponseswhichoccurduringheatstress.Examples

ofsuchresponsesinclude,butarenotlimitedto,sweatingandevaporativeheatloss,alterationsin

skin blood flow and dry heat loss, and alteration in cardiovascular function to support

thermoregulation.

23

2.5.1. Sweatingandevaporativeheatloss

Asambienttemperatureormetabolicheatproductionincreases,thereisincreasingdependence

uponthesudomotorfunctionandevaporativeheatlosstodissipatebodyheat.Therearetypically

twotypesofhumansweatglands,apocrineglandsandeccrineglands(Shibasakietal.,2006).The

eccrinesweatglandareprimarilyresponsibleforthermoregulatorysweatinginhumans(Satoetal.,

1989).

Eccrinesweatglands

Eccrinesweatglandsaredistributedovernearlytheentiresurfaceofthehumanbody.Thenumber

ofhumansweatglandstypicallyrangesfrom1.6to4.0million.Theeccrinesweatglandstructure

consistsofabulboussecretorycoilwhichleadstoaduct.Thissecretorycoilislocatedinthelower

dermisandtheductextendsthroughthedermallayersandopensdirectlyontotheskinsurface.

Theuncoileddimensionofthesecretoryportionoftheeccrinesweatglandisapproximately30-50

µmindiameterand2-5mminlength.Theadultsecretorycoilrangesfrom1to8x10-3

mm3

(Sato

andSato,1983).Thereisapositivecorrelationbetweenthesizeofanindividualsweatglandand

themaximalsweatrate(SWR)ofthatgland(SatoandSato,1983).

Given the challenges of identifying neural tracts in humans, the exact neurological pathways

responsible for thermoregulatory sweating are not entirely understood. Evidence from animal

studiessuggeststhatefferentsignalsfromthepreoptichypothalamustravelviathetegmentumof

theponsandthemedullaryrapheregionstotheintermediolateralcellcolumnofthespinalcord.

In the spinal cord, neurons emerge from the ventral horn, pass through the white ramus

communicans,combinewithperipheralnervesandtraveltosweatglands(Low,2004;Nakamura

etal.,2004).

Sweatrateandevaporativeheatloss

Theinitialincreaseinsweatrate(SWR)duringheatstressisaresultofanincreaseintheactivated

sweatglands;however,additionalincreasesinSWRoccurthroughanincreaseintheproduction

and secretionof sweatpergland (Kondoet al., 2011;Randall, 1946). The recruitmentof sweat

glandsisrapid,withnearmaximalrecruitmentbeingachievedinaslittleas8-minofexerciseand

passiveheatstress.Incontrast,theincreasesinsweatoutputperglandaremoregradual(Kondo

etal.,2011).Theratethatsweatevaporates,isdeterminedbythegradientbetweentheskinand

airvapourpressures,andthecoefficientofevaporativeheattransfer.Thewiderthewatervapour

gradient, the greater the rate of evaporation (Sawka et al., 2011a). Furthermore, warm skin

24

enhancesthesweatresponse(Nadaletal.,1971),whichislikelymediatedbyboththeincreased

localtemperatureandassociatedincreasedskinbloodflow(Wingoetal.,2010).

Heat loss through the evaporation of sweat is an essential process in the thermoregulatory

responsetoexercise.Forexample,whenanindividualperformsexerciseatametabolicrateof600

W,approximately80%oftheenergyconsumedbecomesheat.Assuch,480W(28.8kJ.min−1

)must

bedissipatedtoavoidheatstorage.Thespecificheatofbodytissueapproximates3.5kJ.kg.°C−1

,so

a70kgmanora60kgfemalehasaheatcapacityof245kJ.°C−1

or210kJ.°C−1

,

respectively.Ifsweat

isnotsecreted,Tcincreasesbyapproximately1.0°Cevery8-min30-sforthe70kgmale(245kJ.°C−1

/28.8kJ.min−1

)or7-min20s forthe60kgfemales(210kJ.°C−1

/28.8kJ.min−1

).However,asthe

latent heat of evaporation is 2.43 kJ.g−1

, secretion and evaporation of ∼12 g of sweat (28.8kJ.min

−1

/2.43kJ.g−1

)perminwouldenabletheheattobetransferredtotheenvironmentavoiding

heatstorage(Sawkaetal.,2011a).

Whenperformingexercise inahighambientcondition,humanshave thecapacity toproducea

remarkableamountofsweat.Individualsperformingactivitiesinbothadesertandtropicalclimates

havebeenreportedtoexperienceameanSWRof1.6L.hr-1

whenpartiallyclothed,and2.0L.hr-1

whenwearingfullprotectiveclothing(Montainetal.,1994).Factorssuchasexerciseintensityand

duration,clothing,andenvironmentalconditionsultimatelyaffectanathlete’sSWR.ObservedSWR

inavarietyofathletesduringtrainingandcompetition,rangefrom0.2L.hr-1

to3.4L.hr-1

(Sawkaet

al.,2007).

2.5.2. Skinbloodflowanddryheatloss

In thehumanbody,bloodtransfersheatbyconvection fromthedeepbody tissues to theskin.

Bloodflowtothecutaneouscirculationhasatremendousrange,fromnearlyzeroinextremecold

toapproximately7L.min-1

whenTc ishigh(JohnsonandKellogg,2010).Reflexescontrollingskin

blood flowhave the capacity to use nearly this entire range.When thermoregulatory sweating

begins,skinbloodflowservesasameanstodeliverheattotheskinwhichisthenremovedbysweat

evaporation(Sawkaetal.,2011a).Thus,skinbloodflowandsweatingworktogethertodissipate

theheat.

Vasoconstrictionandvasodilationcontrolofskinbloodblow

SkinbloodflowisultimatelyaffectedbyTsk,whichdirectlyimpactsonthevascularsmoothmuscle.

CutaneouscirculationisaffectedbytheTskandTc,viareflexesoperatingthroughthesympathetic

nervoussystemfromthethermoregulatorycontrolcentres(JohnsonandKellogg,2010).Certain

25

areas of the body, including the palm of the hand, sole of the foot, lips, ears, and nose, are

predominantly innervated by adrenergic vasoconstrictor fibres (Sawka et al., 2011a). The

vasodilatationactivitythatoccursintheseregionsislargelyduetowithdrawingthevasoconstrictor

activity(Tayloretal.,1984).ThereisminimalvasoconstrictoractivitywhenTskexceeds39°Conthe

remaining body regions, including the arms, legs and torso. Active vasodilatation during heat

exposure depends largely on sympathetic innervations (Taylor et al., 1984). Skin blood flow

responses differ between rest and aerobic exercise during heat stress (González-Alonso et al.,

2008).Exercise increasesvasoconstrictoractivitywhich increases the threshold temperature for

vasodilatationandreducesskinbloodflowathighbodytemperaturescomparedtorest(Sawkaet

al.,2011a).Thesemodifiedskinbloodflowresponsesoccurduringexerciseheatstressbecausethe

cardiovascularsystemischallengedtosupportsimultaneouslybothhighskeletalmusclebloodflow

andskinbloodflow.

Coreandskintemperatureeffectsonskinbloodflow

Table 2.1. illustrates the effect of different Tc and Tsk on the skin blood flow requirements to

preserve heat transfer (Kenefick et al., 2007). In the example, if the net heat production is 7.7

kcal.min-1

andTcandTskare38°Cand30°C,respectively,theskinbloodflowrequirementwillbe

~1.1L.min−1

.WhenTskincreasesto34°Cand36°Casaresultofheatstress,theminimalskinblood

flowrequirementtopreservethesameheattransferrateincreasesto~2.2L.min−1

and~4.4L.min−1

,

respectively.However, ifTc rises to39°C theskinblood flowrequirementsare reducedwithout

compromising heat transfer. This example, demonstrates how an elevated Tsk as a result of a

warmer ambient or reduced evaporative cooling, increases skin blood flow requirements. In

addition,theexampledemonstrateshowanelevatedTcmayreduceskinbloodflowrequirements

andcardiovascularstrainassociatedwithexercise-heatstress.

26

Table2.1Estimatedwholebodyskinbloodflowrequirementsduringprolonged,severerunning

exerciseatdifferentbodycoreandskintemperatures.

Netheatproduction Tc

(°C)

Tsk

(°C)

Tc-TskGradient

(°C)

Skinbloodflow

(L.min-1)watts.min-1 kcal.min-1

540 7.7 38 30 8 1.1

540 7.7 38 34 4 2.2

540 7.7 38 36 2 4.4

540 7.7 39 36 3 3.0

Notes:Skinbloodflow:Qs=[((1/C)xh)/(Tc-Tsk)].WhereC=specificheatofblood~0.87kcal.°C.L-1

;h=heat

production;Qs=skinbloodflowTc=coretemperature,Tsk=skintemperature.Netheatproductionestimated

usingabodymassof60kgandrunningvelocityof325m.min-1

(worldclass42kmpace)aftersubtractingfor

work(20%efficiency)and50%dryandevaporativeheatlosses.Adaptedfrom(Keneficketal.,2007).

2.5.3. Cardiovascularsupportofthermoregulation

Duringexerciseheatstress, thecardiovascular system isprimarily required toprovidesufficient

cardiacoutputtoperfuseskeletalmuscleadequatelytosupportmetabolismwhilstsimultaneously

perfusing the skin to support heat loss (Sawka et al., 2011a). These competingmetabolic and

thermoregulatorydemandsaltercardiacfunctionanddistributionofthecardiacoutput(Brothers

et al., 2009a). When ambient temperature increases from 20°C to 36°C whilst exercising at a

moderateintensity(70%maximalO2uptake(VoO2max)),thereisanincreaseinTc,Tsk,forearmblood

flow,andpercentagechangeinPV,whereasstokevolumedecreases(Nadaletal.,1979).Thehigh

skinbloodflowcombinedwiththegreaterreductioninPV,worktoreducevenouspressureand

thus, cardiac filling (Nelson et al., 2010). Furthermore, an increase in heart rate (HR) reduces

diastolicfilingtime,contributingfurthertoareducedstrokevolume(Fritzscheetal.,1999).

27

Skintemperatureandcardiovascularstrain

An increase in Tsk andanarrower Tcto Tsk gradient,which typically occursduringexerciseheat

stress,resultsinagreatercardiovascularstrain.Cheuvrontetal.(2003)demonstratedtheeffectof

analteredTctoTskgradientoncardiovascularstrain.WhenTcremainsconstant(37.5°C),whilstTsk

is increasedfrom32°Cto35°C,HRincreasedbyapproximately26beats.min-1

.Inaddition,when

raisingTskfrom32°Cto36°CHRincreasedbyapproximately49beats.min-1

.Thisevidencesuggests

thatanarrowTctoTskgradientresultsinagreaterstrainonthecardiovascularsystem.

Redistributionofbloodflowwithexerciseheatstress

Duringexerciseheatstress,thecardiovascularsystemsabilitytodeliveradequatebloodsupplyto

theskeletalmuscleandcerebrumcanbecompromised(González-Alonsoetal.,2008;Nyboetal.,

2002;Rasmussenetal.,2010).Although,cardiacoutputissustainedduringlowintensityexercise

in the heat, cardiac output is compromised atmoderate to high intensity exercise in the heat

(Gonzalez-Alonso,2003).Tosupportthedemandsofthecardiovascularsystemduringexerciseheat

stress,bloodisredirectedfromthevisceratotheskinandactiveskeletalmuscles(Sawkaetal.,

2011a).Thevisceralbloodflowreductionsaregradedtotheexerciseintensity,andtheeffectsof

exercise and heat are additive. Secondary to the reduced visceral blood flow, blood can be

mobilizedfromthecompliantsplanchnicbedstohelpmaintaincardiacfillingduringexerciseheat

stress(Sawkaetal.,2011a).

Cardiovascularcomplicationsassociatedwithexerciseheatstress

Thehighdemandsplacedon thecardiovascular systemduringexerciseheat stressmay lead to

syncopeandorthostatic intolerance(KenefickandSawka,2007).Hyperthermiaanddehydration

areoftenrelatedandmaymodifycardiacfilling,bloodpressure,andbaroreceptorreflexregulation,

whichcontributetoorthostaticintolerance(Charkoudianetal.,2003;Crandalletal.,2010;Wilson

etal.,2009).Inaddition,hyperthermiacanreducelocalveno-arteriolarresponses(Brothersetal.,

2009b)andreducecerebralvascularconduction(Wilsonetal.,2006)andaccompanyingcerebral

perfusionduringorthostaticchallenges.

2.6. Sexdifferencesinphysiologicalresponsestoheatstress

Sexhastraditionallybeenconsideredanindependentmodulatoroftemperatureregulation.Ithas

beenwellreportedthatmales’andfemales’thermoregulatoryresponsestoexerciseintheheat

differ(Kaciuba-UscilkoandGrucza,2001;Kenney,1985;Nunneley,1978).Thetwocommonheat

28

lossavenueswhichhavebeencomparedbetweenmalesandfemalesareevaporative(sweating)

anddry(skinbloodflow)heatloss.

2.6.1. Sweatrateandevaporativeheatloss

Females,havepreviouslydemonstratedalowerSWRwhilstmaintainingasimilarTctothatofmales

(Avellinietal.,1980;Keatisuwanetal.,1996).TheprimaryconcernwithcomparingSWRbetween

malesandfemalesisthattypicallyexperimentaldesignsadoptaprotocolusingarelativeVoO2max.

AdministeringexerciseatarelativepercentageofVoO2maxwillresultinsubstantialdifferencesin

absoluteO2consumption(Keatisuwanetal.,1996).Forexample,amaleandfemalewithabody

mass(BM)of70kgand60kgrespectively,bothwitharelativeVoO2maxof50mL.kg-1

.min-1

willboth

havearelativeO2consumptionof25mL.kg-1

.min-1

whenexercisingat50%ofVoO2max.However,

males will have an absolute O2 consumption of 1,750 mL.min-1

and females

1,500 mL.min-1

respectively. This 250 mL.min-1

difference provides a metabolic heat production that is

approximately 85W greater inmales comparedwith females (Gagnon et al., 2008). The lower

resultantrateofheatproductioninfemaleswouldtherefore,havenecessitatedasmalleramount

ofevaporationfromtheskinandsubsequentlyalowersweatproductiontosatisfybiophysicalheat

balance requirements. Thus, the previously reported thermoregulatory differences observed

between males’ and females’ SWR may be confounded by the inherent physical differences

betweenthesexes.

Sexdifferencesintemperatureregulationduringexercisehavebeenshowntoexistirrespectiveof

confoundingdifferencesinmetabolicheatproductionandphysicalcharacteristics.However,these

differencesonlybecomeevidentaboveacertainrequirementforheatlossandaresolelyattributed

toalowersweatglandoutputinfemales(GagnonandKenny,2012).Specifically,femalesexhibita

lowerincreaseinbothlocal(GagnonandKenny,2012)andwholebody(GagnonandKenny,2012,

2011)sudomotoractivityasafunctionofincreasesinTc(i.e.,thermosensitivity),withnodifferences

in the onset threshold of these responses. The lower thermosensitivity of sudomotor activity,

combinedwith a lack of sex differences in the onset threshold suggests that sex differences in

sudomotoractivityduringexercisearemediatedperipherally(GagnonandKenny,2011,2012).

2.6.2. Skinbloodflowanddryheatloss

To date, there is very little data published evaluating the sex differences in the sensitivity of

cutaneous vasodilation using methods that eliminate the inherent limitation associated with

exercise intensityprescribedwhencomparingmalesand females, asdescribed in section2.6.1.

29

Following passive heating, Inoue et al. (2005) reported that females have a relatively greater

cutaneousbloodflowonthethighcomparedwithmales,howevernodifferenceswereobserved

atanyoftheothersites(Inoueetal.,2005).Thehighercutaneousbloodflowwassuggestedto

reflect a greater degree of cutaneous vasodilation on this site, since no sex differences were

observed in themeanarterialpressure(Inoueetal.,2005).Theseresultssuggest, that females’

dependenceuponthecutaneousvasodilationresponseforheatlossisgreaterthanthesweating

response. Furthermore, it was suggested that the regional differences between sexes in

vasodilatationandheatdissipationonthethighmayberelativelygreaterinfemalesthaninmales.

Itislikelythatheatdissipationfromthelimbsismoreeffectivethanheatdissipationfromthetrunk,

becausetheBSA:BMratioofthelimbsisgreater.Conversely,GagnonandKenny(2012)reported

nodifferenceinskinbloodflowresponsesbetweenmalesandfemales,followingexerciseatafixed

requirementofheatloss.TheseobservationsarefurthersupportedbyGagnonetal.(2013)who

reportednodifferencesbetweenmalesandfemales’cutaneousvascularconductancetovarying

dosesofacetylcholineandsodiumnitroprusside.Althoughthereremainsnoconclusiveevaluation

of the skin blood flow differences between males and females, it remains an important

considerationwhencomparingthermoregulatoryresponsesbetweensexes.

2.7. Menstrualcycle

Themenstrualcycleisaseriesofvarioushormonereleasesthatcoordinatethereadinessofthe

femalereproductivesystemforconception(figure2.8).Atypicalmenstrualcycleoccursovera28-

dperiodandisbrokenupintotwophases;thefollicularphase(D1–D13)andthelutealphase(D15

–D28).Othercommonterminologymaybeusedtodescribethedifferentmenstrualcyclephases

(table2.2).Ondayoneofthemenstrualcycle,thehypothalamusreleasesgonadotropin-releasing

hormonewhichcontrolsstimulationandinhibitionofthereleaseoffollicular-stimulatinghormone

(FSH)andluteinisinghormone(LT)fromtheanteriorpituitary.Thesehormonesthenregulatethe

release of oestrogen from the ovarian follicles and progesterone from the corpus luteum,

coordinatingtheprogressionoftheseeventsduringanaveragecycleof28-d(MarshandJenkins,

2002)(figure2.8)

An essential consideration to make when conducting physiological research within a female

populationisthetimingoftestinginrelationtothemenstrualcycle.Thefluctuationsinhormones

associatedwiththemenstrualcyclemayalterthefunctionofanumberofdifferentphysiological

responses associated with thermoregulation later discussed in section 2.9. The most common

methodtominimisethechangesinhormoneconcentrationsassociatedwiththemenstrualcycle,

thatmayinfluencethedependentvariable,istostudyfemalesinthesamephaseoftheirmenstrual

30

cycleacrossparticipants,oracrossstudydaysinlongitudinalstudies.Femalesaremostcommonly

tested during the early follicular phase (D1-D7), when both oestrogen and progesterone

concentrationsareattheirlowestlevels.

Despitetheconvenienceofstudyingfemalesintheearlyfollicularphaseofthemenstrualcycle,the

broaderclinicalrelevanceofthefindingsmaybelimitedsincefemalesareinthispartoftheircycle

foronly~25%oftheirreproductivelives.Although,oestrogenandprogesteroneconcentrationsare

lowrelative tootherphasesof thecycle,hormone levels remainconsiderablyhighercompared

withthoseofmen.Thus,theirimpactwhenmakingcomparisonsbetweenthesexesisnotentirely

eliminated.Researcherscanattempttokeepthecycledaysconsistentacrossfemaleswithinagiven

studytoinvestigatethephysiologicaleffectsindependentofcyclephase,buttheymusttakeinto

account the variability of these hormones across cycle phases within females. Additional

considerationsforhormoneverificationincludethelargevariabilitybetweenindividualswithinthe

same menstrual cycle phase, the diurnal variation in hormone secretion with progesterone

concentrations typically higher in themorning and the known effect of exercise on increasing

oestrogenandprogesteroneconcentrations(SyropandHammond,1987).

31

Figure2.8Typicalcoretemperature(Tc)andhormonechangesacrossthemenstrualcycle.Noteall

valuesareapproximatevaluessincetheyvarygreatlyacrossindividuals.LH=luteinizinghormone

FSH=folliclestimulatinghormone.AdaptedfromStachenfeld&Taylor,(2014).

32

Table2.2Commonterminologyandhormoneconcentrationsassociatedwithatypical28-dcycle

withovulationoccurringonD14.

Notes:AdaptedfromJansedeJonge,(2003).

2.8. Verificationofthemenstrualcyclephase

Researchersadoptavarietyofmethodstoverifythemenstrualcyclephase.Thesemethodsinclude

countingdaysfromtheonsetofmenses,dailyrecordingsofbasalTc,measuringLHconcentrations

for the predictionof ovulation, and themost establishedmethod, ofmeasuringoestrogen and

progesteroneconcentrationinthebloodplasmaorbloodserum.

2.8.1. Countingdaysfromtheonsetofmenses

A well established, commonly used method to determine the menstrual cycle phase, involves

femalescountingthedaysfromtheonsetofmenses.Thismethodassumesthatindividualshavea

regular menstrual cycle that can be applied proportionally to an average menstrual cycle

representation.However,activewomenwithregularbleeding,havebeenreportedtohaveahigh

incidenceoflutealphasedeficientcyclesandanovulation(DeSouzeetal.,1998).Thus,counting

daysfrommensesdoesnotdifferentiateovulatoryfromanovulationandmayprovideinaccurate

Menstrualcyclephase

(daysofmenstrualcycle)

Concentrations Additionalterminology

(dayofmenstrualcycle)Estradiol Progesterone

EarlyFollicular(2-7)

Low

Low

Follicular(1-13)

PreOvulatory(1-13)

MidFollicular(6-9)

LateFollicular(9-13)

High

Low

MidCycle(12-18)

Ovulatory(3-5daroundovulation)

Ovulation(14)

MidLuteal(18-24)

High

High

Luteal(15-28)

PostOvulatory(15-28)

33

information regarding the menstrual cycle phase. Furthermore, assumptions are made that

individualscorrectlyreporttheirmenstrualactivity.

2.8.2. Basalbodytemperature

Anothermethod formenstrual cycle determination is daily recording of basal Tc. This method

assumesaregularpatternofactivitythroughoutthecycle.Typically,ovulatorywomenhavea0.3°C

increase in basal Tc subsequent to ovulation, which is sustained throughout the luteal phase

(HorvathandDrinkwater,1982).However, therelationshipbetweenbasalTcandovulationmay

varyconsiderably,sincesomefemalesdonotexperienceanincreaseinbasalTcduringtheluteal

phase(Bauman,1981).Although,anincreaseinbasalTcoftenreflectsanincreaseinprogesterone,

aweakcorrelation(r≤0.302)haspreviouslybeenfound(Formanetal.,1987).Thus,thereliability

ofrecordingdailybasalTctodeterminemenstrualcyclephasesanditsreflectionofprogesterone

levelsshouldbetreatedwithcaution.

2.8.3. Urinaryluteinisinghormoneconcentration

Urinary LH concentration can be determined using ovulation predictor kits, consisting of

colorimetricenzymeimmunoassaysofurinaryLH(JansedeJonge,2003).WhentheurinaryLHsurge

hasbeenshown,itcanbeassumedthatovulationwilltakeplacewithinthenext14-26hr(Miller

andSoules,1996).

2.8.4. Measurementofoestrogenandprogesterone

OnD1ofthemenstrualcyclegonadotropin-releasinghormone issecretedbythehypothalamus

which controls the stimulation and inhibition of FSH and LH (Marsh and Jenkins, 2002). These

hormones regulate the releaseofoestrogenandprogesteroneduringanaveragecycleof28d.

Consequently, the measurement of oestrogen and progesterone can be used to verify the

menstrual cycle phase. The hormone concentrations can be measured in blood serum, blood

plasmaandsaliva.Inaddition,theirmetabolitescanbemeasuredinurine.Measuringoestrogen

and progesterone concentration in blood serum or blood plasma confirms themenstrual cycle

phase,basedonanincreaseinprogesteronefromthefolliculartothelutealphase,indicatingthat

ovulationhasoccurred(JansedeJonge,2003).

34

2.9. Thermoregulatorydifferencesoverthemenstrualcycle

Inwomenwhohavenosignofanymenstrualcycleirregularity,themenstrualcyclecomprisesa

follicularphaseandalutealphaseduringwhichoestrogenandprogesteroneconcentrationsalter.

ChangesintheconcentrationsofoestrogenandprogesteronemodifythebasalTcandthethreshold

forsweatingandcutaneousvasodilationbyalteringtheactivityofcentralregulatorymechanisms

(CharkoudianandJohnson,2000).Specifically,theelevationofprogesteroneconcentrationduring

the lutealphase iscorrelatedwithan increase inthebasalTc (0.3-0.5°C)andthethresholdfor

sweatingandcutaneousvasodilation(CarpenterandNunneley,1988;CharkoudianandJohnson,

2000; Inoue et al., 2005). Whereas, the elevation of oestrogen concentration during the late

follicularphasecausesadecreaseinthesameparameters(StephensonandKolka,1999).Themost

widelyacceptedexplanationforanelevatedTcinthelutealphaseisthatthethermoregulatoryset

point is increased (Forman et al., 1987). Thus, this would imply that the threshold for all

thermoregulatory effector responses are shifted in a similar direction and the increased luteal

phasebasalTcwouldremainelevatedthroughoutexerciseand/orheatstress.

Themechanismsthatcontrolsthesealterationsinthethermoregulatorysetpointassociatedwith

the menstrual cycle are not well understood in females. In animal research, progesterone

administrationhasbeenshowntodecreasetheactivityofthewarmsensitiveneuronsandincrease

theactivityofcoldsensitiveneuronsinthepreopticarea(Nakayamaetal.,1975).Thesefindings

indicateacentraleffectofprogesteroneinthepreopticarea,resultinginanincreasedsetpoint

temperature. In contrast, a decrease in Tc has been associated with oestrogen administration

(Tankersley et al., 1992). Animal research indicates that oestrogen increases the activity of the

warmsensitiveneuronsinthepreopticarea(SilvaandBoulant,1986)causingadecreaseinbody

temperature(SilvaandBoulant,1986).Asaresult,researchershavespeculatedthattheincreased

thermoregulatorysetpointduringthelutealphase,isrelatedtotheratiobetweenoestrogenand

progesterone(Formanetal.,1987).

Alterationsinthethermoregulatorysetpointandeffectorresponsesoverthemenstrualcyclehave

beenexaminedinfemalesusingexerciseand/orheatstress.Someresearchershavereportedno

differences in basal Tc between the follicular and luteal phase (Avellini et al., 1980;Wells and

Horvath,1974).Incontrast,someresearchershavereporteddifferencesinbasalTc,howeverthese

differenceswere not apparent following exercise and/ or heat stress (Horvath andDrinkwater,

1982;Senay,1973).Themajorityofthesestudiesfailedtoverifythemenstrualcyclephasewith

measurements of oestrogen and progesterone concentrations accurately. However, when

hormonalverificationof themenstrual cyclehadbeenconducted, the reduction inbasalTchas

35

been reported to remain elevated throughout exercise and/ or heat stress (Carpenter and

Nunneley,1988;HessemerandBruck,1985;Stachenfeldetal.,2000).Severalofthesestudiesalso

reportedanincreasedTcthresholdforthermoregulatoryeffectorresponsesduringthemid-luteal

phase(HessemerandBruck,1985;Stachenfeldetal.,2000).Accumulatively,thesefindingssupport

theincreasedthermoregulatorysetpointtheory.

2.9.1. Sweatingandskinbloodflow

Itiswellestablished,thatthethresholdforonsetofsweatingisincreasedduringthelutealphase

relative to the follicular phase (Hessemer and Bruck, 1985; Stephenson and Kolka, 1985). In

addition,anincreaseinwholebodySWRhasbeenobservedduringthelutealphasecomparedwith

the follicular phase (Grucza et al., 1993; Hessemer and Bruck, 1985). However, during intense

exercise in hot environmental conditions (35°C and 50°C), where there is a greater strain on

evaporativeheatloss,nodifferencesinSWRhavebeenobservedbetweenthefollicularandluteal

phases (Kolka and Stephenson, 1989; Stephenson and Kolka, 1985). The potentialmechanisms

responsiblefortheincreasedthresholdfortheonsetofsweatingduringthelutealphasecouldbe

associatedwiththeactionsofeitherprogesteroneoroestrogen,asbothareelevatedduringthis

time.Thestimulustoincreasesweatproductionoccurscentrally,adirectresultofprogesterone

actingonthepreoptic/anteriorhypothalamus(StephensonandKolka,1993).

The fluctuations inhormonal concentrationofoestrogenandprogesteroneassociatedwith the

menstrualcyclehavebeenshowntoalteractivecutaneousvasodilatorycontrols(Bruntetal.,2012;

Charkoudian and Johnson, 2000; Charkoudian, 2010). When oestrogen and progesterone

concentrations are elevated during the luteal phase, there is a concurrent increase in the Tc

thresholdforcutaneousvasodilation,relativetothefollicularphase(Charkoudian,2001).However,

beyond the threshold for cutaneous vasodilation, further increases do not incur additional

alterations (Charkoudian, 2001). Horvath and Drinkwater (1982) reported a 25% reduction in

forearmbloodflowatrestduringexposuretoambientconditionsof28°Cduringthefollicularphase

comparedwiththelutealphase.However,whenexercisewasperformedinbothawarm(35°C)

and hot (48°C) environment therewere no differences in forearmblood flowbetweenphases.

Similarthresholdshiftsoccurwithadministrationoforalcontraceptivesthatcontainoestrogenand

progesterone (Charkoudian and Johnson, 2000; Grucza et al., 1993; Stachenfeld et al., 2000;

StephensonandKolka,1993).Thisobservedshiftinthresholdsistheneteffectofareductionin

oestrogenandanelevationofprogesterone,thelatterhavingdominance.Theimpactofoestrogen

andprogesteroneonsweatingandskinbloodflowresponsesarefairlyconclusive.Consequently,

36

consideration of themenstrual cycle phasemust bemadewhen assessing these variables in a

femalepopulation.

2.9.2. Cardiovascular

Onlyafewalterationsincardiovascularresponseshavebeenreportedasaresultofthechangesin

hormoneconcentrationsduringthemenstrualcycle.AnelevationinrestingandexercisingHRhas

previouslybeenreportedduringthelutealphaseofthemenstrualcyclecomparedtothefollicular

phase.Kolka&Stephenson, (1997) reportedan increase inHRwhenexercising at 80%ofpeak

aerobicpowerin35°Cand22%RH,inthemidlutealphase(161±9beats.min-1

)comparedtothe

earlyfollicularphase(150±11beats.min-1

).ThisevidenceisfurthersupportedbytheworkofInoue

et al., (2005) who reported higher resting and exercising HR during the luteal phase (80 ± 4

beats.min-1

, 100 ± 5 beats.min-1

) compared to the follicular phase (70 ± 3 beats.min-1

, 93 ± 5

beats.min-1

)ofthemenstrualcycle.However,Tenaglia,McLellan,&Klentrou,(1999)reportedno

phaserelateddifferencesinHRresponsesduringa300-minexposureinvolving15-minintervalsof

walkingandrestin40°Cand30%RH.Evidenceremainsinconclusiveregardingthemenstrualcycle

effectonHRresponsesatrestandduringexerciseinhotconditions.

It remains unclear whether PV is affected by menstrual cycle related fluctuations in hormone

concentrations.Chapmanetal.(1997)reportednodifferencesinPVbetweenthefollicular(45±3

mL.kg-1

)and the lutealphase (47±3mL.kg-1

)of themenstrual cycle. In contrast,Calzoneetal.

(2001)reportedalowerPVinthelutealphase(2437±113mL)comparedtothefollicularphase

(2529±183mL). Furthermore, thedifferencesobservedmaybedue to theprocedureused to

determinePV;Chapmanetal., (1997)usedamodifiedcarbonmonoxide rebreathing technique

whichdirectlymeasuresbloodvolumeandCalzoneetal.,(2001)usedadyedilutiontechniquewith

Evans bluewhich directlymeasures PV. A lower PV in the luteal phase is further supportedby

Stachenfeldetal.(1999)whoreporteda8.4±2.5%higherPVinthefollicularphasecomparedto

thelutealphase.Thealterationsarepotentiallyduetotheincreasedlevelsofoestrogenassociated

withthe lutealphaseof themenstrualcycle.An increase inoestrogen isknowntoenhancethe

actionsofaldosteroneintheabsorptionofsodiumintherenaltubules(Stachenfeldetal.,1998)

causingareductioninPV.

Generally, no differences are observed in haemoglobin concentrations over the course of the

menstrual cycle (Lebrun et al., 1995;Maughan et al., 1996; Tenaglia et al., 1999). In contrast,

Jurkowskietal.(1981)reportedanincreaseinrestinghaemoglobinduringthelutealphaseofthe

menstrualcycle,althoughthisdidnotresult inan increase inarterialO2content,orO2delivery

37

during exercise. Furthermore, haematocrit levels generally remain unchanged both at rest and

duringexerciseoverthecourseofthemenstrualcycle(Chungetal.,1999;Tenagliaetal.,1999).

However, Stachenfeld et al. (2000) reported an increase inhaematocrit levels during the luteal

phase(39.3±0.5%)comparedtothefollicularphase(38.3±0.7%).Evidenceremainsinconclusive

as to whether consideration of the menstrual cycle phase must be made when investigating

females’cardiovascularandhaematologicalresponsesacrossthemenstrualcycle.

2.9.3. Exerciseperformanceintheheat

Although,femaleathletestrainandcompleteduringallphasesoftheirmenstrualcycle,itisofhigh

importancetounderstandtheeffectsthatthehormonalfluctuationsassociatedwiththemenstrual

cyclemayhaveonexerciseperformance.TheincreasesinTctypicallyobservedduringtheluteal

phaseofthemenstrualcyclemayresultinanincreasedthermoregulatoryandcardiovascularstrain

causing,adecreaseinprolongedexerciseperformance.

Tenaglia et al. (1999) examined the effects of the possible thermoregulatory changes over the

menstrual cycle on exercise time to exhaustion in the heat. A longer time to exhaustion was

reported in the early follicular phase during light intensity intermittent exercise. Tenaglia et al.

(1999) also reported a higher Tc during rest in the luteal phase, however, no differenceswere

observedintherateofTcriseortheTcattheendofthetimetoexhaustion.Thesefindingsassume

thatthereisacriticalTclimitforexerciseperformance,thus,duringthelutealphasewhenresting

Tciselevated,exercisetimetoexhaustionwillbelimited.However,thereremainslimitedliterature

conducted within this area. Future research is warranted to determine whether the

thermoregulatory differences across the course of themenstrual have on effect on endurance

performance.

VoO2max isa strongpredictorofenduranceperformance.Data indicates that there isnophase

relateddifferencesinVoO2orVoO2maxinhotconditions(HorvathandDrinkwater,1982;Stachenfeld

etal.,2000;Tenagliaetal.,1999).However,Williams&Krahenbuhl(1997)reportedhigherresting

VoO2 inthelutealphase(4.8±0.1L.min-1

)comparedtothefollicularphase(3.9±0.2L.min-1

). In

contrast,Lebrunetal.(1995)reportedalowerVoO2maxinthelutealphase(53±1mL.kg-1

.min-1

)

comparedtothefollicularphase(54±1mL.kg-1

.min-1

).WilliamsandKrahenbuhl(1997)reported

lowerventilationrateduringthefollicularphase(10.3±0.8L.min-1

)comparedtothelutealphase

(12.4±0.7L.min-1

)whilstatrest.Furthermore,ahigherventilationratewhilstexercisingat55%

VoO2maxand80%VoO2maxduringthefollicular(42.2±1.4L.min-1

;63.6±2.0L.min-1

)comparedto

thelutealphase(46.2±0.9L.min-1

;68.8±3.0L.min-1

)ofthemenstrualcyclewasalsoreported.An

38

increaseinprogesteroneconcentrationsduringthelutealphaseofthemenstrualcycleincreases

thephrenicnerveactivity.Asthephrenicnerveinnervatesthediaphragm,elevatedprogesterone

duringthelutealphaseisoftenassociatedwithvaryingdegreesofhyperventilation(Denisetal.,

1976).Toconclude,althoughprogesteronehasbeenreportedtohaveamarkedeffectonphrenic

nerve activity, subsequent influences of the menstrual cycle on respiratory function are not

conclusive.Thus,considerationofthemenstrualcyclephasesshouldbeconsideredwhenassessing

thermoregulatoryresponsesinfemales,asalterationsinVoO2willultimatelyaffectthemetabolic

heatproduction.

2.10. Individualdifferenceinheattolerance

Individuals vary in their ability to sustain heat strain, with some demonstrating a decreased

capability todissipateheatandgreaterbodyheatcontentunder thesameexerciseheatstress.

Theseindividualshavebeendescribedasheatintolerantandareoftencharacterisedbyanearlier

andgreater rise inTc, agreater storageofmetabolicheat,haveahigherphysiological strain to

moderateintensityexerciseintheheatandreducedsweatingsensitivity(Epstein,Shapiro,&Shai,

1983;Moran,Heled,Still,Laor,&Shapiro,2004).Aheatintolerancestatecanbeeithertemporary

or permanent. It may stem from transient predisposing factors such as an acute injury to the

thermoregulatorycentre,insufficientHA,dehydration,orinfectiousdisease.Inaddition,alasting

thermoregulatorydysfunctionmay stem fromconditions such as cardiacdisease, diabetes, and

stokeimpairmenttosweatglands(Epstein,1990),orduetodifferencesingeneexpression(Moran

etal.,2006).Congenitalfactorssuchasectodermaldysplasiamayalsocompromiseheattolerance1

insomeindividuals.Forthepastseveraldecades,researchershaveundertakeninvestigationsto

develop a better understanding of the various factors that contribute to the inter-individual

variationsobserved in thehumanheatstress response.Historically,anthropometryandaerobic

fitnesshavebeenconsideredtobetheprimarytransientdeterminantsthatalterthermoregulatory

responses.

2.10.1. Aerobicfitness

Traditionally, itwasbelievedthatTcchangesduringexercisewerecloselyrelatedtotherelative

exerciseintensity(SaltinandHermansen,1966).Tobalancetheincreaseinmetabolicheat,higher

1

Thetermheattolerancewillbeusedthroughoutthisthesistodescribethephenotypicresponses

toheatstress,whereasthetermthermotolerancewillbeusedtodescribethecellularresponsesto

heatstress.

39

aerobically trained individuals would typically increase their heat loss, resulting in similar Tc

responsetountrainedindividuals.Inanattempttoisolatetheinfluenceoftheconfoundingeffects

of aerobic capacity between groups, many studies over the past few decades comparing

thermoregulatory responses between independent groups have consequently administered

exerciseusingpercentageVoO2max.

Mora-Rodriguezetal.(2010)reportedthatwhenexerciseisadministeredrelativetoVoO2max(40%

and60%VoO2max),aerobicallytrainedindividuals(66±6mL.kg-1

.min-1

)experiencesimilarchanges

in Tc to untrained individuals (44 ± 3 mL.kg-1

.min-1

). Enhanced heat dissipation in the trained

participantspermittedsimilarTcchangescomparedtotheuntrainedparticipants,despiteahigher

netmetabolicheatproduction.However,whenexercisingatahigherintensity(i.e.80%VoO2max)

Tcwashigherintrainedindividualscomparedwithuntrainedindividuals.Thus,thesuperiorheat

dissipation of the trained participants did not compensate for the higher net metabolic heat

productionwhenexercisingatahigherpercentageofVoO2max.

Itiswidelyacknowledged,thatthehumanthermoregulatorysystemworkstoattainasteadystate

Tc,suchthattherateofmetabolicheatproductionisbalancedbyanequallyelevatedrateofheat

dissipation (Jay and Kenny, 2007). The increase in heat production during exercise is mostly

determined by the change in absolute rate of O2 consumption, whereas the increase in heat

dissipationoccursmainlyduetoanelevatedrateofsweatevaporationfromtheskin(Gagnonet

al.,2013).Thus,whenexercisingatasimilarpercentageofVoO2max,endurancetrainedparticipants

exerciseatahigherabsoluteworkratethanuntrainedindividualsandgeneratemoremetabolic

heat (Gagnonet al., 2008). In turn, cutaneousblood flow (Fritzsche andCoyle, 2000) and SWR

(Kuwaharaetal.,2005)arehigherintheaerobicallytrainedparticipantscomparedtotheuntrained

participantstocounterbalancetheincreasedheatproduction.

Cramer&Jay(2014)reportedthattherearenodifferencesinwholebodySWRbetweenaerobically

fitindividuals(60mL.kg-1

.min-1

)andrelativelyunfitindividuals(40mL.kg-1

.min-1

)duringexerciseat

a fixed heat production of 275 W.m-2

, despite large differences in percentage VoO2max.

Furthermore, the only fitness-related differences in local SWRwere observed on the forehead,

wheresweatingwaslowerinindividualswithagreateraerobiccapacity.Thisfindingwaspotentially

duetogreatersympatho-adrenalactivityassociatedwithagreatercardiovascular,perceptualand

ventilatorystraininthelessfitindividualsatafixedheatproduction,potentiallyelicitingagreater

sweatproductioninglaborousskinareas(Crameretal.,2012).Thisevidencesuggeststhatprevious

observations that have used relative VoO2max to assess the influence of aerobic fitness on

thermoregulatoryresponses,maybeconfoundedbymethodologicalissues.Thus,whenisolating

40

theindependentinfluenceofphysiologicalfactorsonthethermoregulatoryresponsestoexercise

using a between subject design, exercise intensity should be administered using fixed heat

production.

2.10.2. Anthropometry

AhighbodyfatpercentagemayalterTcchangesduetoaloweraveragespecificheatcapacityof

adiposetissue. Jayetal. (2011)comparedthethermoregulatory responsesof individualswitha

high(60±5mL.kg-1

.min-1

)andlow(40±3mL.kg-1

.min-1

)aerobiccapacity,whilstexercisingatafixed

heatproduction.Theparticipantsinthelowaerobiccapacitygrouphadatwofoldhigherbodyfat

percentage(22.2±7.0%)comparedwiththehighaerobiccapacitygroup(11.9±5.9%).Thelower

specificheatcapacityofadiposetissue(2.97kJ.-1

kg-1

.°C)wouldhaveyieldedaslightlyloweraverage

specificheatofthebody inthe lowaerobiccapacitygroup(3.53kJ.-1

kg-1

.°C)relativetothehigh

aerobicgroup(3.45kJ.-1

kg-1

.°C)(GeddesandBaker,1967).Therefore,asimilarbodyheatstorage

wouldhavetheoreticallyelevatedTcinthelowgroupbyagreatermagnitude.Nevertheless,the

changesinTcwerealmostidenticalbetweenthetwogroups(high:0.87±0.15°C,low:0.87±0.18°C)

when exercising at a fixed heat production. Therefore, it appears that any thermoregulatory

implicationofthedifferenceinadipositybetweenthetwogroupswasinsignificant,likelydueto

any insulation created from the fat layers beingbypassedbyblood flow following theonsetof

exercise (Havenith and Middendorp, 1990). Another implication of a higher adiposity is a

concomitant difference in body volumeand thereforeBSA. The lower density of fat (0.9 kg.L-1

)

relativetomuscle(1.1kg.L-1

)willallowforagreaterevaporativerateinthelowaerobiccapacity

groupduetothehigherbodyfatpercentage.

Cramer&Jay(2014)comparedthethermoregulatoryresponsesofparticipantswithasmall(68±6

kg)andlarge(92±7kg)BMwhilstexercisingatafixedrateofmetabolicheatproduction(500W

and600W).Resultsdemonstratethat largedifferencesinBMsystematicallyalteredTcchanges,

with smaller participants achieving a greater Tc rise. However, when exercise intensity was

prescribedusingafixedheatproductionrelativetoBM(6.5W.kg-1

and9.0W.kg-1

)thesystematic

differencesinTcwereeliminateddespitedifferencesinBM,absoluteheatproduction,andrelative

exercise intensity.Thus, thehigherTc in thesmallergroupat thesame fixedheatproduction is

directlyexplainedbytheinfluenceofBMperseandnotbyanydifferencesinheatdissipationor

bodycomposition.Whenexercisingataheatproductionofboth500Wand600W,nodifferences

inTskandtherefore,dryheatlosswereobservedbetweenparticipantswithasmallandlargeBM.

Thisresultedinasimilarabsoluteevaporativerequirementandtherefore,thesamewholebody

SWRandpresumablyevaporation(CramerandJay,2014).Assuch,itislikelythatthedifferences

41

inbodyfatpercentagebetweenthetwogroupscontributedtotheobserveddifferences inTre.

WhileitmaybepossiblethatmuchlargerdifferencesinbodyfatpercentagealterchangesinTc,

theindependentinfluenceofhighversuslowadiposetissuewhilstcontrollingforheatproduction

and BM has not yet been evaluated and merits further investigation. Thus, when assessing

thermoregulatory responses to exercise, using a between subject design with participants of

varyingbodysizes,exerciseintensityshouldbeadministeredusingfixedheatproduction.

2.11. Methodstoassessheattolerance

ExperimentalprocedureshavebeenemployedtoraiseTcunderrestingandexerciseconditionsto

challengethethermoregulatoryresponsesasamethodofassessingthermoregulatorymechanisms

(Inoueetal.,2005;Kenney&Hodgson,1987;Montain,Sawka,Cadarette,Quigley,&McKay,1994).

AHTTcanprovideinformationtoevaluatetheabilitytoavoidtheriskofexertionalheatstrokeand

aid in decisions about readiness to return to training, competition and/or active duty. The

importance of heat tolerance screening was highlighted by Schutte (2010). Following the

implementationofheattolerancescreening,incidencesofheatstrokeandheatillnessinmining

werereducedto33casesovera2yr.periodcomparedwith88fortheprevious3yrs.(Schutte,

2010).

Exercise HTTs commonly adopt protocols of a long duration (>100 min), low intensity (≤ 50%

VoO2max)inenvironmentalconditionsof35-40°Cand40-50%RH,usingavarietyofexercisemodes

including,runningandbenchstepping(Epstein,1990;Moranetal.,2007).Theextensivevariation

intestparadigmsmakesbetweenstudycomparisonsdifficult.Constructvalidityoftheseprotocols

has been assumed since physiological measures were different between groups of individuals,

althoughtheprotocolsemployedwerenotalwayscapableofdifferentiatingbetweenheattolerant

andheatintolerantindividuals(Moranetal.,2004).Shapiroandcolleagues(1979)developedaHTT

whichconsistedofsteppingfor180-minona30cmhighbenchinacontrolledhotenvironment.

This was adapted by the IDF to be performed on a treadmill. The protocol involves 120-min

exposureto40°Cand40%RHwhilewalkingonatreadmillatapaceof5km.hr-1

anda2%elevation

(Moran et al., 2007). Criteria to discern tolerance and intolerance to exercise heat stresswere

developed.Attheendofthe2-hrexposure,heattolerancewasdefinedasapeakTre≤38.0°C,peak

HR≤120beats·min-1

,andSWR≥780g·h-1

.Heatintolerancewasdefinedas,peakTre≥38.5°Cor

peakHR≥145beats·min-1

(Moranetal.,2007)andapeakphysiologicalstrainindex≥6(Moranet

al.,2004).

42

ThereareseverallimitationsassociatedwithIDFHTT,whicharemoreapparentwhenconducting

thetestonanathleticpopulation(Johnsonetal.,2013).Endurancerunnerstypicallyrunathigher

intensitiesforshorterdurationsthanmilitarypersonnel.TheintensityoftheprotocoloftheHTTis

considerablybelowthatatwhichathleteswouldtrainandcompete.Thus,theHTTmisrepresents

the metabolic heat production endurance runners may experience and potentially may

misdiagnosetheirsusceptibilitytoahyperthermicstate,orworseaheat-relatedillness.Protocols

ofthisdurationwouldrequireasignificantamountoftimeoutfromtrainingforathletes,which

mayresultinacoachand/orathletebeingreluctanttohavetheirheattoleranceassessed.Further

work is required to establish an effective short duration, running based HTT improving the

ecologicalvalidityandapplicationtoendurancerunners.

2.12. Exertionalheatillnessesinrunning

The challenges of training and competing in a hot environment are complex and difficult to

comprehendfully,sinceathletesareaffectedinavarietyofwaysduringhighintensityexercisein

hothumidenvironments.Pathologiceventswhichoccur inhotenvironmentsare referredtoas

exertionalheatillnessesandareoftencategorisedasexertionalheatstroke,heatexhaustionand

heatcramps(table2.3).Exertionalheatillnessescommonlyaffectathletesduringhighintensity,or

long duration exercise and the severity of the illness depends largely upon the degree of

hyperthermiaanditsduration.Ourknowledge,regardingexertionalheatstrokeislimitedtocase

studies of athletes who push beyond the normal physiological limits, since laboratory studies

cannotbeconducted,duetotherisksofseverehyperthermiaareethicallyunacceptableforhuman

research. The survival of these athletes depends largely on prompt recognition and the

implementation of effective cooling strategies to limit tissue exposure due to destructive

hyperthermia(HoweandBoden,2007).

43

Table2.3Heatillnessesandtheirassociatedsymptoms.

Condition Tc(°C) AssociatedSymptoms

Heatedema Normal None.

Heatrash Normal Pruriticrash.

Heatsyncope Normal Dizziness,generalweakness.

Heatcramps Normalorelevated

<40

Painful muscle contractions, affected muscles

firmtopalpate

Heatexhaustion 37-40 Lowbloodpressure,elevatedpulseand

respiratoryrates,sweatyandpale,headache,

weakness,dizziness,nausea,vomiting,

diarrhoea,irritabilityanddecreasedmuscle

coordination.

Exertionalheatstroke ≥40 Disorientation,confusion,dizziness,irritability,

headache,lossofbalance,andmusclefunction,

profoundfatigue,hyperventilation,vomiting,

diarrhoea,delirium,seizuresorcoma.

Notes:Coretemperature,Tc.AdaptedfromHoweandBoden,(2007).

Ahighincidenceofexertionalheatillnesshasbeenreportedinlongdistancerunners,with31%and

53%ofthetotalcasesofexertionalheatillnessesduringthe1992NewOrleansU.S.OlympicTrials

and the 1996 Atlanta Olympics respectively, occurring in long distance runners (Martin, 1997).

Furthermore, during the 2004 BostonMarathonwhere ambient temperature averaged 22.5°C,

morethan300emergencymedicalcallswereobserved,despitechangestotheracestarttimefrom

1200-hrto1000-hrinordertodecreaseheatstressandrelatedcasualties(Roberts,2010).The2007

LondonMarathonexperiencedhigherambienttemperaturesthanexpected,withanaverageair

temperatureof19°C,versusanaverageof12°C(ElHelouetal.,2012).DuringtheLondonmarathon

in2007,73hospitalisationswererecordedwithsixcasesofsevereelectrolyteimbalanceandone

44

death.Incontrast,thenumberofpeopletreatedwas20%lowerattheLondonMarathonayear

laterin2008,whenconditionswerecoolandrainywithanaverageambienttemperatureof10°C

(Roberts,2010).Furthermore,thetotalaveragetimetocompletethemarathonwas17-minslower

in2007comparedtopreviousyears. Inaddition, thepercentageofracewithdrawals inChicago

2007was thehighest (30.74%)amongall60marathonracesanalysedbyElHelouetal., (2012)

when temperature were 13°C higher than the average temperature. Sixty six runners were

admittedtothehospitalwith12reportedtobeintensivecarecaseswithhydrationdisorders,heat

shocksyndromesandonedeath.

Ina12yr. summaryofmarathonmedicalencounters, therewere1.2casesofheatcrampsper

1,000 race entrants; accounting for 6.1% ofmedical encounters (Roberts, 2000). Furthermore,

duringa14kmroadraceinmildconditions(11°C-20°C),14heatexhaustioncaseswerereported

in10,000finishers(RichardsandRichards,1984).Exertionalheatstrokehasbeenobservedmost

commonly during activities that involve continuous high intensity exercise. The twin cities

marathonwhichisrunincoolconditionsaveragesoneexertionalheatstrokepatientper10,000

finishers (Roberts, 2000). Furthermore, during an 11.5 km road race perform in hot humid

conditions there were approximately 10-20 exertional heat stroke cases per 10,000 finishers

(Brodeuretal.,1989).Despitethevolumeofrunnersexperiencinganexertionalheatillnessduring

runningevents,therearecurrentlynoevidencebasedrecommendationsregardingthereturnof

anathletetotrainingandcompetition.TheAmericanCollegeofSportMedicine(HoweandBoden,

2007)recommendthatexertionalheatstrokecasualtiesreturntotrainingandcompetitionwhen

they have re-established heat tolerance. However, there is currently no establishedmethod to

evaluatearunner’sheattolerancetodeterminewhethertheyhavere-establishedheattolerance.

Establishingsuchaprotocolmaypreventathletesfromreturningtotrainingandcompetitiontoo

soonandreducethelikelihoodofexperiencinganotherepisodeofaheatrelatedillness.

2.13. Heatacclimation

Humansareregulators,andassuchstrivetokeepmeanbodytemperaturewithinanarrowrange

(37.0±0.5°C).Accordingly,humansnotonlypossess thecapabilityofenduringawiderangeof

climatic stressors, but also have a considerable capacity to extend this tolerance, through

adaptation.Thephysiologicaladaptationsthatoccurduringexposuretotheheatarereferredtoas

acclimatization responseswhen seen in a naturally occurring environment andHA responses if

observed ina controlledenvironmental setting.HA isachievedbyapersistent challenge to the

thermoregulatorycontrolsystemthatimprovesheatdissipationandthermotolerance,prolonging

endurancetoheatstress.

45

Heatacclimationdevelopsthroughfrequentexposuretohotenvironmentalconditions,whichelicit

responsesthatattenuatethenegativeeffectsofheatstress.HAadaptationarediscussedindetail

insection2.15.Inshort,thebenefitsofHAincludeanimprovedthermoregulatory,cardiovascular,

sweatingandskinbloodflow,cellular,andmetabolicfunctioning,areductioninperceptualstrain

andanimprovedexerciseperformance(Horowitz,2014;Périardetal.,2015;Sawkaetal.,2011a;

Taylor,2014)(table2.4.).ThetimecourseofHAistypicallycategorizedintoSTHA(≤8d)andLTHA

(≥10d)(Garrettetal.,2011;Guyetal.,2015;Périardetal.,2015).Themagnitudeofphysiological

adaptation inducedbyHAdepends largelyonthe intensity,duration,frequency,andnumberof

heatexposures(Sawkaetal.,2011b).

2.14. Heatacclimationprotocols

Heat acclimation is typically achieved through one of three induction pathways; fixed intensity

(Castleetal.,2011;Lorenzoetal.,2010),self-pacedexercise(Armstrongetal.,1986;Sunderlandet

al.,2008)orcontrolledhyperthermia(Gibson,etal.,2015;Pattersonetal.,2004a,2014;Garrettet

al.,2009,2012;Reganetal.,1996).Although,exerciseintheheatisthemosteffectivemethodfor

developingHA,passiveheatexposurehasalsobeenshowntoincursomeadaptation(Beaudinet

al.,2009;Scoonetal.,2007;Stanleyetal.,2015;Takamataetal.,2001).

2.14.1. Fixedintensityheatacclimation

Traditionally,HAaimedtopreparepeopletoperformaspecifictaskwithinaknownsetofclimatic

conditions.Thisinvolvedrepeatedlyapplyingafixedendogenousandexogenousthermalloadto

increaseTcasaresultofanimbalancebetweenheatproductionandheatloss(Castleetal.,2011;

Houmardetal.,1990;Kresfelderetal.,2006;Lorenzoetal.,2010;Sandströmetal.,2008).Thus,

duringeachHA sessionparticipants’workat the sameabsoluteexercise intensity for the same

duration.Thismodeldevelopshabituation, reducesphysiologicalstrain,and increases tolerance

(Foxetal.,1963a).Whilethisapproachappearslogical,therearemajorlimitationsregardingthe

effectivenessandinterpretationofthisprocedure.Itscapacitytoinduceadaptationcontinuallywill

progressivelydeclineasadaptationoccurs,sincethestrengthoftheadaptationstimulusisreduced.

In addition, these protocols require individuals to work at the same absolute intensity; the

physiological strain they experience will therefore, be variable since individuals differ in their

inherent heat tolerance (Taylor, 2014). When undertaking mechanistic research, the resulting

variationsinphysiologicalstrainduringadaptationmakedatainterpretationchallenging.

46

2.14.2. Self-regulatedheatacclimation

Self-regulatedexercise requiresparticipants to followprescribedworkand rest intervals,whilst

self-selecting and modifying the work rate. Although, this method has considerable practical

applicationsinceitcanbeusedtorehearsepacingstrategiesorsimulategameplay,thetechnique

has no standardised endogenous thermal loading, reducing control and possibly constraining

adaptation(TaylorandCotter,2006).

2.14.3. Passiveheatacclimation

Passive HA uses exogenous heat to incur thermoregulatory stress, with a minimal metabolic

contribution.Adaptationisinducedifthecumulativeadaptationimpulseissufficientlylarge,and

thiscanbebroughtabout throughhot-waterbathing (TurkandThomas,1975;Zurawlewetal.,

2015)andbypassiveexposure toanenvironmental chamber, steamroomor sauna (Foxetal.,

1963a; Scoon et al., 2007; Stanley et al., 2015). While passive techniques are beneficial, the

techniquehasbeenreportedtobe lesseffectivethanexercise intheheat (Shapiroetal.,1981;

Wyndham, 1973). The reason is that passive heating may only induce a slight-to-moderate

homeostaticdisturbance,sothecumulativeadaptationimpulseremainssmall.

Foxandcolleaguesdevelopedaveryeffectivepassivetechniquetosolvethislimitation(Foxetal.,

1963a). The method was based on elevating and clamping Tc (controlled hyperthermia).

Participantswereheated in a hot (43°C), humid (100%RH) condition causing a rapid rise in Tc.

Participantsweredressed invapour-barrier suits thatwere continuously ventilatedwithhotair

(35°C)andenteredasecondchamber(38°C),wheretheyrested.ThreeclampedTc(37.3°C,37.9°C,

and38.5°C)andthreeexposuredurations(30,60,and120-min)wereevaluatedfor12-d.Results

suggesteda60-minclampat38.5°Cwas required toestablish fullHA (Foxetal.,1963a).These

specificationshaveledtouseofcontrolledhyperthermiaduringexercise.

2.14.4. Controlledhyperthermiaheatacclimation

ControlledhyperthermiaHAensuresequalthermalstrainisplaceduponparticipantsasitinvolves

elevatingandmaintaininga steadystatebody temperatureabove thesweating thresholdusing

exercise(Garrettetal.,2011).TheuseofthecontrolledhyperthermiaHAhasbecomeincreasingly

popular. Thismethod has consistently been found to reduce thermal strain and increasework

capacityduringbothSTHAandLTHAandmayoffermorecompleteadaptationtofixedintensityHA

(Nealetal.,2015;Gibsonetal.,2015;Garrettetal.,2009,2012;Pattersonetal.,2004a;Reganet

al.,1994).

47

2.15. Heatacclimationinduction

Humanshavearemarkableabilitytoadapttoheatstressandgivenadequatewaterandprotection

fromthesun,ahealthyheatacclimatedindividualcantolerateextendedexposuretovirtuallyany

natural,weather-relatedheatstress.HAresultsinaseriesofadaptationsthatreducetheharmful

effectsofheatstress.Typically,adaptationoccursthroughmorphological,chemical,functional,and

genetic adjustments that decrease physiological strain under stress. HA involves a number of

complexadaptations(table2.4),whichallintegratetoenhanceexerciseperformance.

2.15.1. Sweatingandskinbloodflow

SudomotoradaptationswereamongthefirstdescribedinresponsetoHA.Bytheendofthe1940s,

it was widely accepted that HA increased SWR and decreased sweat sodium and chloride

concentrations(Dilletal.,1933).Theobservedshiftintheonsetthresholdforsweating(Patterson

etal.,2004a)andanalteredSWR(table2.5)andcomposition,areconsideredtheprincipaladaptive

responsestoheatexposure(Buonoetal.,2009a).Theseresponsesareindicativeofbothcentral

andperipheraladaptation.Atthecentrallevel,HAdecreasestheTcatwhichsweatingisinitiated.

Thisadjustmentinonsetthresholdisproposedtocorrespondtoanabsolutechangeinmeanbody

temperature,ratherthantotheattainmentofapredeterminedmeanbodytemperature(Patterson

etal.,2004a).Peripheraladaptations,manifestedbychangesinSWRandsensitivityandoccurat

thelevelofthesweatgland(Buonoetal.,2009a;Inoueetal.,1999).FollowingSTHAandLTHAusing

acontrolledhyperthermiamodel,thereisonaveragea0.23L.hr-1

and0.46L.hr-1

increaseinSWR

observed(table2.5).Thepotentialmechanismsthatexplainthesechangesinclude,anincreased

cholinergicsensitivityoftheeccrinesweatglandandincreaseglandularhypertrophyandefficiency

(Lorenzo&Minson,2010;Buonoetal.,2009;Sato&Sato,1983).Inaddition,eccrinesweatglands

becomeresistanttohidromeiosis,resultingintheabilitytosustainahigherSWRforaprolonged

periodoftime(Foxetal.,1963b).

InadditiontoanenhancedSWR,sweatcompositionchangeswithHA.Electrolytesarereabsorbed

andsweatsodiumconcentrationisreduced,resultinginamoredilutesweat(Chinevereetal.,2008;

Saatetal.,2005).Thepreservationofsodiumappearstostemfromincreasedsodiumconservation

withinthere-absorptiveductofthesweatgland(SatoandDobson,1970;Satoetal.,1971),which

is dependent on aldosterone, a hormone secreted in response to exercise andheat stress that

facilitatesthereabsorptionofsodium.EvaporativeheatdissipationisenhancedforagivenSWR

whenamoredilutesweatissecreted,sinceitismoreeasilyevaporatedbecauseofawideningof

thewatervapourgradientbetweentheskinandambientair(Taylor,2014).

48

Table2.4Summaryofadaptationstoheatacclimation.

AdaptationOutcome

Thermoregulatory Tc(restandexercise) Reduced

Tsk Reduced

Cardiovascular HR Reduced

Strokevolume Increased

Cardiacoutput Bettersustained

Totalbodywater Increased

PV Increased

Bloodpressure Betterdefended

Myocardialcompliance Increased

Myocardialefficiency Increased

Cardio-protection Improved

Sweatingandskinbloodflow Sweating(earlieronsetandhigherrate) Improved

Electrolytelosses Reduced

Skinbloodflow(earlieronsetandhigherrate) Improved

Cellular HSP Increased

Acquiredthermotolerance Increased

Metabolic Wholebodymetabolicrate Lower

Muscleglycogen Spared

Perceptual Thirst Moresensitive

RPE Improved

TS Moreaccurate

Performance Lactatethreshold Increased

Maximalaerobicpower Increased

Submaximalaerobicperformance Improved

Skeletalmuscleforcegeneration Increased

Notes:HR,heartrate;HSP,heatshockprotein;PV,plasmavolume;RPE,ratingofperceivedexertion;Tc,core

temperature;TS,thermalsensation;Tsk,skintemperature.AdaptedfromSawkaetal.,(2011a).

49

AnearlierandgreatersudomotorresponseduringHAimprovesevaporativecooling,assumingthe

climateallowsevaporation,andreducesTskandthus,skinbloodflowrequirements.AlowerTskmay

alsoreducecutaneousvenouscompliance,suchthatthebloodvolumeisredistributedfromthe

peripheraltothecentralcirculation(Rowelletal.,1967).Untilrecently, itwasassumedthatHA

centrally modifies thermoregulatory responses in the skin by reducing the Tc threshold for

vasodilation,withoutalteringtheslopeofthebloodflow–Tcrelationship(Foxetal.,1963a;Roberts

et al., 1977; Yamazaki and Hamasaki, 2003). However, by locally infusing an endothelium-

dependentvasodilator (acetylcholine)viamicrodialysis, Lorenzo&Minson (2010)demonstrated

thatHAimproveslocalcutaneousvascularresponses.Theauthorssuggestedthatthisperipheral

responsemaybeassociatedwithanincreaseinthenumberandsensitivityofmuscarinicreceptors,

adecreaseincholinesteraseactivity,leadingtoanimprovedvascularresponsetoacetylcholine,or

alterations inthepathwayofvasodilationwithinsmoothmusclesortheendothelialcells.Given

thatHAdoesnotaltermaximalskinbloodflow,themodifiedcutaneousvascularresponseappears

to stem from improvements in vascular function (i.e., increased sensitivity of the skin

microvasculature to vasodilate), rather than structural changes that limit maximal vasodilator

capacity(LorenzoandMinson,2010).

50

Table2.5Changeinwholebodysweatrateobservedfollowingshort-andlong-termcontrolledhyperthermiaheatacclimation.

Citation No.ofParticipants Sex V9O2peak(mL.kg-1.min-1) No.ofsessions Exposure(min) Temp(°C) RH(%) ∆SWR(L.hr-1)

STHA≤7d

(Gibsonetal.,2015) 8 M 49 5 90 40 39 0.34

(Nealetal.,2015) 10 M 63 5 90 40 50 0.13

(Pattersonetal.,2004a) 11 M 54 6 90 40 60 0.22

Mean±SD 10±2 55±7 5±1 90±0 40±0 50±11 0.23±0.11

LTHA≥10d

(Gibson,etal.2015) 8 M 49 10 90 40 39 0.56

(Gibsonetal.2015) 8 M 57 10 90 40 41 0.54

(Pattersonetal.,2004a) 11 M 54 16 90 40 60 0.29

(Pattersonetal.,2014) 8 M 54 17 90 40 59 0.44

Mean±SD 9±2 54±3 13±4 90±0 40±0 50±11 0.46±0.12

Notes:LTHA,long-termheatacclimation;M,male;RH,relativehumidity;STHA,short-termheatacclimation;SWR,sweatrate;Temp,ambienttemperature;VVO2Peak,peakoxygenuptake.

51

2.15.2. Bloodvolumeandfluidbalance

Heatacclimationhasbeenreportedtoincreasetotalbodywaterbyapproximately2–3L,(Basset

al.,1955;Pattersonetal.,2014,2004b).Thisincreaseiswellwithinthemeasurementresolution

(4.9%) for total body water (Maw et al., 1996) and thus, appears to be a real physiological

phenomenon.Thechangesinintracellularfluidandextracellularfluidthatcontributetowardsthe

increaseintotalbodywaterarevariable;sinceextracellularfluidhasbeenreportedtoaccountfor

greater,equal,andsmallerthanitspercentageincreaseintotalbodywaterafterHA(Sawkaand

Coyle,1999).Thereisrelativelyhighvariabilityassociatedwiththemeasurementofextracellular

fluid,andtherefore,trendsforsmallchangesaredifficulttointerpret.Thechangestointracellular

fluidaretypicallycalculatedasthedifferencebetweentotalbodywaterandextracellularfluid,and

thus resultsareunclear since themeasurementvariability inherent inboth these techniques, is

confounded in thecalculationof intracellular fluid.The increase in secretionof fluidconserving

hormones aldosterone and arginine vasopressin, and/or renal sensitivity to a given plasma

concentrationinpartexplaintheincreaseintotalbodywater(Périardetal.,2015).Furthermore,

theconservationofsodiumassistsinthemaintenanceofosmolalityintheextracellularfluidwhich

maintainsorincreasesextracellularfluidvolumeduringheatadaptation(Noseetal.,1988).Asa

result of an increase in total body water and extracellular fluid following HA, youmay expect

expansionofPV.

Following3-4dofheatexposureorasaresultofseasonalchanges,PVexpansionispresent(Sawka

andCoyle,1999).A5%expansioninrestingPVoccursinthehottestmonthsoftheyearanda3%

contraction in the coldestmonths (Sawka and Coyle, 1999). However, individuals have varying

responses,withsomeindividualsnotexperiencinganyexpansioninPV.TheobservedPVexpansion

following HA has previously been described as a transient phenomenon (Shapiro et al., 1981),

howeverthismayhavebeenanexperimentalartefactrelatedtothetraditionalconstantworkrate

modelofHA.Recentfindings,suggestthatbyusingthecontrolledhyperthermiatechnique,which

maintainsa constantadaptationstimulusbyclampingTc throughoutHA,PV iselevatedby15%

following10consecutiveexposures(Gibsonetal.2015)andremainssimilarlyexpanded(∼14%)after8-22dofheatexposure(Pattersonetal.,2004a,2014).Followingcontrolledhyperthermia

HA,PVexpansionisapproximately7%and14%forSTHAandLTHA,respectively(table2.6).The

magnitudeof increase inPV is typicallydependentupon theHAday, thehydration statewhen

measured,Tskandwhethertheindividualisatrestorperformingexercise(Harrison,1985;Kenefick

et al., 2014; Sawka et al., 1983). The magnitude of PV expansion may also depend on the

participants’ fitness level, since endurance training will typically result in an expanded PV. PV

52

expansion offers a thermoregulatory advantage, since it increases vascular filling to support

cardiovascularstability(Sawkaetal.,2011b).

Areductionintotalbodywaterasaresultofdehydrationwilladverselyaffectthermoregulation

andincreasecardiovascularstrain(SawkaandCoyle,1999).Thus,dehydrationwillcounteractthe

adaptationstothermoregulationinitiatedfollowingHAandaerobictraining(Sawkaetal.,1983);

whichthemoreseverethelevelofdehydration,thegreaterthestrainandthegreaterelevationin

Tc.Dehydrationalsoimpairsdryandevaporativeheatlossviathedevelopmentofplasmahyper-

osmolalityandhypovolemia(SawkaandCoyle,1999).However,Takamataetal. (2001)provides

evidencetosuggestthatHAmightattenuatetheadverseeffectsofhyper-osmolalityonimpairing

sweatingandskinbloodflowresponses.Assuch,totalbodywaterexpansionstemmingfromHA

mayconferaprotectivebenefitagainstdehydration.Althoughitisapparentthatdehydrationhas

clear detrimental effects on exercise performance, moderate permissive dehydration during

exerciseHAmayacceleratetheHAprocess,(TaylorandCotter,2006)byincreasingfluidelectrolyte

retention,PVexpansion,andcardiovascularresponsestoheatstress(Garrettetal.,2014,2011;

Nealetal.,2015).

53

Table2.6Changeinplasmavolumeobservedfollowingshort-andlong-termcontrolledhyperthermiaheatacclimation.

Citation No.ofParticipants Sex V9O2peak(mL.kg-1.min-1) No.ofsessions Exposure(min) Temp(°C) RH(%) ∆PV(%)

STHA≤7d

(Garrettetal.,2012) 8 M 66 5 90 40 60 4.5

(Garrettetal.,2014) 9 M 60 5 90 40 60 4.0

(Nealetal.,2015) 10 M 63 5 90 40 50 0.0

(Pattersonetal.,2004b) 12 M 54 6 90 40 59 15.0

(Pattersonetal.,2014) 8 M 54 5 90 40 59 13.1

Mean±SD 9±2 59±5 5±1 90±0 40±0 58±4 7±6

LTHA≥10d

(Gibson,etal.,2015) 8 M 57 10 90 40 41 15.0

(Pattersonetal.,2004b) 12 M 54 16 90 40 59 14.4

(Pattersonetal.,2014) 8 M 54 17 90 40 59 12.8

Mean±SD 10±2 55±2 14±4 90±0 40±0 53±10 14±1

Notes:LTHA,long-termheatacclimation;M,male;PV,plasmavolume;RH,relativehumidity;STHA,short-termheatacclimation;Temp,ambienttemperature;V9O2Peak,peakoxygen

uptake.

54

Eichnaetal.(1950)demonstratedthatHAreducesvoluntarydehydrationasaresultofanimproved

relationshipbetweenthirstandbodywaterneeds.Consequently,heat-acclimatedindividualsare

betterabletomaintainhydrationduringexerciseintheheat,andthusminimizebodywaterdeficits

and voluntary dehydration, provided that access to fluids is not restricted. This is an essential

adaptation since following HA SWR increases and if fluid replacement is not proportionately

increased,greaterdehydrationwilloccur.AlongwiththeexpansionofPVinducedduringHA,the

ability to better maintain fluid balance through thirst represents an adaptive response that

contributestoreducecardiovascularstrainduringsubsequentheatstress.

2.15.3. Cardiovascularstability

Following STHA and LTHA using a controlled hyperthermia model, resting HR reduces by 3

beats.min-1and12beats.min-1respectively(table2.7).Thereareseveralpotentialmechanismsthat

explainthereductionincardiovascularstrain.Thesemechanismsinclude,animprovedskincooling

andredistributionofbloodvolume,PVexpansion, increasedvenousreturnfromcutaneousand

non-cutaneousvascularbedsandreducedTskandTc(table2.8).

FollowingHA,centralcardiacfunctionwasassessedbyeitherwalkingat5.6km.h−1(Rowelletal.,

1967) or cycling at 50% of their maximal aerobic power (Nielsen et al., 1993) until volitional

exhaustion.Duetotheprotocoladministeredtheheatstrainwasvariablebetweensessionsand

between the pre andpostHAheat stress tests.Nevertheless, both groups observed significant

elevationsinstrokevolumealongwithreducedHRfollowingHA.Thesechangessignifyanimproved

cardiacfunctionsinceHRandTcwerelower(Nielsenetal.,1993;Rowelletal.,1967).Wyndham,

(1951)andWyndhametal.,(1976,1968)findingsaredivergenttothatobservedbyRowelletal.

(1967)andNielsenetal.(1993).ThesestudiesalladoptedafixedintensityHAprotocolandthus,

theabsolute stress levelwas identicalbetween thepreandpostheat stress test. FollowingHA

cardiacoutputandHRdeclined,whilestrokevolumeremainedconstant.Theseobservationsmay

inpartbeexplainedbythereductionintheHAstimulustowardstheendofHA,asaresultonthe

fixedintensityprotocol.However,Wyndhametal.(1976)reporteda25%increaseincardiacoutput

following4–7dofHA,howeverthiswasonly8%following10d.Theobservedchangestocardiac

outputmayinpartbemediatedbyPVexpansionwhichwasreportedinacompanionpaperusing

thesameparticipants(Senayetal.,1976).Inthispaperitwasreportedthattherewasagradual

elevationinPVoverthefirst6-dofHA,afterwhichitremainedstable.Interestingly,nochangesin

HR,strokevolumeorcardiacoutputwerereportedduringsubsequentmaximalexerciseintheheat

(40°C,30%RH)following10-dHAintrainedcyclists(Lorenzoetal.,2010).However,whenthesame

55

exercise was performed in cool environment (13°C, 30% RH), both stroke volume and cardiac

outputweresignificantlyhigher.

Gotoetal.(2010)reportedrestingstrokevolumetobeelevatedfollowingHA.Thisispotentially

mediatedbyabettermaintenanceofventricularfillingpressure(GledhillandJamnik,1994),and

blood volume, which support venous return and cardiac preloading. Indeed, during times of

multisystemstress,asmallPVelevationcandictateone’sabilitytoaccommodatethesestresses

and to avoidhypotension and cardiovascular insufficiency.Workingwith a ratmodel,Horowitz

(2003)hasadvancedourunderstandingintothecardiovascularadaptationachievedfollowingHA.

Whilethetranslationofthesemodificationstohumansisspeculativeatpresent,theyhelpexplain

someadaptationspreviouslyexplained.There isrightwardshift inthediastolicpressure-volume

relationship of the heart following chronic heat exposure (Horowitz et al., 1986b), signifying

increased cardiac compliance. This is important, since it indicates the heart can be filledmore

readily and without relying on the relatively small blood volume elevation accompanying heat

adaptation to increase central venous pressure. This observed change in compliance enhances

strokevolume,making theheartmoreeconomicaland lowering themetaboliccostofpumping

(Horowitz et al., 1986b). The corresponding increase in efficiency reduces metabolic heat

production. Finally, since there is an increase in myocardial contractile performance following

endurancetraining(SchaibleandScheuer,1979)andHAtypicallyinvolvesenduranceexercise,then

overallcardiacfunctionappearstobeenhanced.

56

Table2.7Changeinrestingheartrateobservedfollowingshort-andlong-termcontrolledhyperthermiaheatacclimation.

Citation No.ofParticipants Sex V9O2peak(mL.kg-1.min-1) No.ofsessions Exposure(min) Temp(°C) RH(%) ∆HRrest(beats.min-1)

STHA≤7d

(Garrettetal.,2012) 8 M 66 5 90 40 60 -4

(Garrettetal.,2014) 9 M 60 5 90 40 60 0

(Gibsonetal.2015) 8 M 49 5 90 40 39 -5

(Pattersonetal.,2004a) 11 M 54 6 90 40 60 -5

Mean±SD 9±1 57±7 5±1 90±0 40±0 55±10 -3±2

LTHA≥10d

(Gibsonetal.2015) 8 M 49 10 90 40 39 -8

(Gibsonetal.2015) 8 M 57 10 90 40 41 -18

(Pattersonetal.,2004a) 11 M 54 16 90 40 60 -10

Mean±SD 9±2 56±2 12±5 90±0 40±0 47±12 -12±5

Notes:LTHA,long-termheatacclimation;M,males;RH,relativehumidity;STHA,short-termheatacclimation;HRrest,restingheartrate;Temp,ambienttemperature;VTO2Peak,peakoxygen

uptake.

57

Table2.8Changeinrestingcoretemperatureobservedfollowingshort-andlong-termcontrolledhyperthermiaheatacclimation.

Citation No.of

Participants

Sex V9O2Peak(mL.kg-1.min-1) No.ofsessions Exposure(min) Temp(°C) RH(%) ∆Tcrest

(°C)

STHA≤7d

(Garrettetal.,2009) 10 M 56 5 90 40 60 0.0(Garrettetal.,2012) 8 M 66 5 90 40 60 -0.1(Garrettetal.,2014) 9 M 60 5 90 40 60 -0.2(Gibsonetal.,2015) 8 M 49 5 90 40 39 -0.1(Nealetal.,2015) 10 M 63 5 90 40 50 -0.2

(Pattersonetal.,2004a) 11 M 54 6 90 40 60 -0.2(Pattersonetal.,2014) 8 M 54 5 90 40 59 -0.2

Mean±SD 9±1 57±6 5±0 90±0 40±0 55±8 -0.1±0.1

LTHA≥10d

(Foxetal.,1963a) 18 M - 10 120 43 100 -0.2(Gibsonetal.,2015) 8 M 49 10 90 40 39 -0.1(Gibson,etal.,2015) 8 M 57 10 90 40 41 -0.5

(Pattersonetal.,2004a) 11 M 54 16 90 40 60 -0.3(Pattersonetal.,2014) 8 M 54 17 90 40 59 -0.3

(Welleretal.,2007) 16 M 51 10 110 46 17 -0.3Mean±SD 12±5 53±3 12±3 98±13 42±3 52±28 -0.3±0.1

Notes:LTHA,long-termheatacclimation;M,males;RH,relativehumidity;STHA,short-termheatacclimation;Tcrest,restingcoretemperature;Temp,ambienttemperature;VTO2Peak,

peakoxygenuptake.

58

2.15.4. Thermotolerance

Thermotoleranceoracquiredcellularthermotoleranceisthenomenclatureusedtodescribethe

cellular adaptation caused by a single, or repeated severe, but non-lethal heat exposure that

preconditionsanorganismtosurviveasubsequentandotherwiselethalheatstress(Kuennenet

al.,2011;Moseley,1997).IthasbeensuggestedthatthermaltoleranceandHAshareacommon

basis,asbothmaypotentiallybegovernedbytheheatshockresponse(HSR)(Kuennenetal.,2011).

Assuch, thermal toleranceandHAarecomplimentary,asHAreducesheatstrainandtolerance

increases survival toagivenheat strain. Forexample,Maloyanetal. (1999)demonstrated that

rodents,withfullydevelopedthermotolerance,survive60%moreheatstrainthanwhatwouldhave

been initially lethal. Thermal tolerance is associatedwithheat shockproteins (HSP)2 binding to

denaturedornascentcellularpolypeptidesandprovidingprotectionandacceleratingrepairfrom

heat stress, as well as fever, hypoxia, ischemia, viral infection, energy depletion, and acidosis

(Kregel,2002).HSParegroupedintofamiliesbasedupontheirmolecularmass(8to110kDa),with

HSP72beingparticularlyresponsivetoheatstressandexercise(Locke,1997).Heatshockprotein

families have different cellular locations and functions. At the intracellular (iHSP) level, HSP’s

process stress-denaturedproteins,manageprotein fragments,maintain structuralproteins,and

chaperoneotherproteinsacrosscellmembranes.Intheextracellular(eHSP)milieu,itissuggested

thatHSPactasasignal,triggeringanimmuno-stimulatoryresponse(Pockley,2003).

Heatshockproteinexpressionincreasesduringandfollowingexposuretoheatstresswithdiffering

responses across various tissues (e.g., brain and liver exhibit a greater response than skeletal

muscle).Aftertheinitialexposure,HspmRNAlevelspeakwithinanhourandsubsequentprotein

synthesisdependsuponboththeseverityandcumulativeheatstress(Maloyanetal.,1999).Passive

heat exposure and physical exercise both elicit HSP synthesis (Febbraio and Koukoulas, 2000);

however, the combination of exercise and heat exposure elicits a greater response than either

stressorindependently(Skidmoreetal.,1995).Duringexerciseintheheat,theexpressionofeHsp

hasbeenshowntobebothdurationandintensitydependent,relatingtothelevelofhyperthermia

attainedandrateofriseinTc(Périardetal.,2012).

2The termheat shock protein (HSP)will be used throughout this thesis to describe the proteinexpression whereas heat shock protein mRNA (Hsp mRNA) will be used to describe the geneexpression.

59

Horowitz&Robinson(2007)reportedthatHAincreasesHSP70reservesandacceleratestheHSRin

animals.Inhumans,theHSRduringHAandtheconcomitantexpressionofiHspandeHspremain

somewhatunclear.Following2-dexerciseheatexposureareducedbasallevelofeHsp72andan

increaseexpressionimmediatelypost-exercisehasbeenreported(Marshalletal.,2006).However,

ithasalsobeenshownthatiHSP72isunaffectedoverthesametimecourse(Marshalletal.,2007).

Yamadaetal.(2007)demonstratedthatwhenHAwasextendedto10d,basaliHSP72expression

increased, which blunted the post-exercise induction response. In contrast, eHSP72 remained

unchanged.During a 15-dHA regimen, basal eHSP72 progressively increased and post-exercise

expressiondecreasedina29-yr.oldmaleultra-marathonrunnerpreparingfortheMarathondes

Sables,suggestingthatalongerHAperiodmayinducegreatercellularadaptations(Sandströmet

al.,2008).McClungetal.(2008)conductedafurtherstudyinwhichiHSP72andiHSP90responses

to10-dofexerciseHAwerecorrelatedwithphysiologicaladaptations.SuchthatHAincreasedbasal

levels of both iHSP72 and iHSP90 and individuals who demonstrate the greatest physiological

adaptation exhibit a reduced post-exercise expression (measured ex vivo via water bath

incubation).11-dofcontrolledhyperthermia(1.0°CTcelevation)HAresultedinanincreaseinbasal

iHSP72levels,whileeHSP72expressionremainedunchanged(Magalhãesetal.,2010).Similarto

the findings of McClung et al. (2008), Magalhães et al. (2010) reported a blunted expression

followingexerciseheatexposure inboth iHSP72andeHSP72.Thus, itappearsthat iHSPmaybe

moresensitivetoheatstressthaneHSPandanincreaseinbasallevelduringHAresultsinablunting

of theacute response toexerciseasHAdevelops.ThemeasurementofHsp72mRNA,offersan

alternative marker of the magnitude of the cellular stress response required for increased

thermotolerance,allowingforthedeterminationofwheretheinhibitionhasoccurred.Gibsonet

al.(2015)reportedequalsessionalHsp72mRNAtranscriptionacrossa10-dHAusingbothfixedand

controlledhyperthermia (figure2.9)protocols.TheequalHsp72mRNAincreasesoccurringafter

equal,reducedorincreasedTcfollowingboth5and10-dHA,suggestthataslongasaminimum

endogenouscriterionissurpassed,additionalendogenousthermoregulatorystrainisnotoffurther

benefit,noriscontinualexerciseloadcrucialsolongashyperthermiaispresent.

60

Figure2.9Hsp72mRNApre-andpost-sessionsonD1,D5andD10ofcontrolledhyperthermiaHA

inmaleparticipants.Adaptedfrom(Gibsonetal.,2015a).

Notes:ValuesareMean±SD.*denotessignificantpretopostdifferencewithinsession(p≤0.05).

2.15.5. Exerciseperformance

Heatacclimation isreportedto improveexerciseperformance intheheat.Racinaisetal. (2015)

reportedanaveragetimefora43-kmtimetrialof66±3minand77±6minincool(~8°C)andhot

conditions(~37°C),respectively.Following14-dheatexposuretheaveragetimetrialperformance

was66±4mininahotenvironment,similartothatobservedinacoolenvironmentpriortoHA.

Accordingly,HAmediatedanimprovedsubmaximalexerciseperformancebyreducingphysiological

strain and lessening a variety of other potential fatigue mechanisms (Nybo et al., 2014).

Interestingly,relativetovaluesrecordedintemperateconditions,heatstressmediatesareduction

inVgO2maxintrainedindividualsthatcannotbeabatedbyHA.Lorenzoetal.(2010)reporteda5%

improvementinVgO2maxinacoolenvironment(13°C)and8%improvementinahotenvironment

(38°C)following10-dfixedintensityHA(figure2.10).TheimprovementinVgO2maxobservedina

hotenvironmentfailedtocompensateforthe20%reductioninVgO2maxconferredbyheatstress

before and after HA. Furthermore, therewas a 6% and 8% improvement in 60-min time trial

performanceinacool(13°C)andhot(38°C)environment,respectively(Lorenzoetal.,2010).The

observedimprovementsinVgO2maxweresimilartotheimprovementsobservedinthetimetrial,

reinforcingthenotionthatrelativeexerciseintensitystronglyinfluencesperformanceintheheat

(Périard,2013;Périardetal.,2011).TheimprovementsLorenzoetal.(2010)observedcoincided

0

1

2

3

4

5

6

7

8

9

D1 D5 D10

Hsp7

2mRN

A(Foldchange)

Day

Pre

Post

***

61

withanincreaseinmaximalcardiacoutputandlactatethreshold,PVexpansion,lowerTsk,anda

largercore-to-skingradientfollowingHA.

Interestingly, HA has been reported to have no influence on themaximal Tc an individual can

tolerateduringexerciseintheheat;sinceexhaustioncoincidedwithaTcof∼40.0°ConeachdayofHA,despiteexercisedurationincreasingthroughouttheHAperiod(Nielsenetal.,1993).However,

Robinson(1963)reportedthathighlytrainedrunnersmayreachTcof41.0°Cduringa3milerun(14

min,15s)in30°Cconditions.Furthermore,runnersperformingan8kmrunningtimetrialinwarm

conditions(WBGT=27°C)areabletosustainrunningvelocity,despiteaTcexceeding40.0°C(Elyet

al.,2009).Morerecently,itwasshownthattrainedcyclistsreachTcof40.0°Cattheendofa43.3

km time trial in hot (37°C) conditions (Racinais et al., 2015). Therefore, it appears that aerobic

fitnessconfersanincreasedcapacitytotoleratehigherTc.However,whetherHAprovidesasimilar

benefitremainstobedetermined.

Figure2.10EffectofheatacclimationonVgO2maxinacool(13°C)andhot(38°C)environmentin

maleparticipants.

Notes:Valuesaremeans±standarderrorpreandpost10-dheatacclimation(N=12)andcontroltraining(N

=8).*denotessignificantdifference topreacclimationwithinenvironmentalcondition.Data taken from

(Lorenzoetal.,2010)

50

55

60

65

70

75

Cool Hot Cool Hot

VO₂m

ax(m

l.kg⁻¹.m

in⁻¹)

ExperimentalGroupControlGroup

PreHA

PostHA

**

62

2.15.6. Perceptualresponses

Areduction in thermal sensation (TS) ispotentiallybeneficial forperformance in theheat since

thermal discomfort drives true behavioural thermoregulation (Flouris, 2011). Initiation of these

responsepathwaysduringexerciseelicitsbehaviouralresponseswhichreduceworkrate(Tucker

etal.,2006,2004).Thisisundesirablewithregardstooptimalperformanceintheheat.However,

HAisreportedtoreduceTSduringexercise,performedatasetintensityintheheat.Gibsonetal.,

(2015b)reportedasignificantreductioninbothmeanandpeakTSduringa30-minfixedintensity

heatstresstestfollowing10-d,butnot5-d,ofcontrolledhyperthermiaHA.Theseobservationsare

supportedbyGarrettetal.,(2012)whoobservednochangesinthermalcomfortfollowing5-dof

controlledhyperthermiaHA.Incontrast,Nealetal.,(2015)reportedparticipantshadanimproved

thermalcomfortfollowing5-dcontrolledhyperthermiaHAduringa20-minfixedintensitycycling

intemperateconditions(22°C,60%).TheroleofTSorthermalcomfortareyettobefullyelucidated

withregardstoHA.

2.16. Timecourseofheatacclimation

Heat acclimation is a relatively rapid process that begins on the first day of exposure to a hot

climate.Duringthefirst4-7dofexposure75-80%oftheadaptationsoccur(ArmstrongandMaresh,

1991;Pandolf,1998).TheArmstrongandMareshtableforplateaudaysofphysiologicaladaptation

duringheatacclimatisation(ArmstrongandMaresh,1991)hasevolvedtobeatemporalpatterning

modelofHAacquisition(table2.9).Duringtheinitialheatexposure,physiologicalstrainishigh,as

demonstratedbyanelevatedTcandHR.However,thephysiologicalstraininducedbyheatstress

progressively decreases each day of exposure (Buono et al., 2009a; Houmard et al., 1990).

Cardiovascularfunctionadaptsmostrapidlyin4–5dandvirtuallycompletefollowingsevendaily

exposures(Gibsonetal.,2015;Fujiietal.,2012;Garrettetal.,2009).Inaddition,majorityofthe

thermoregulatoryresponsesincludingreductionsinTskandTctypicallyoccurduringSTHA(Gibson

etal.,2015;Fujiietal.,2012;Garrettetal.,2009).TheexpansionofPV,alongwiththermoregulatory

cardiovascular and metabolic adaptations, improves the perception of effort in athletes.

Traditionally, STHA has failed to provide a strong-enough stimulus for sudomotor adaptation

(Cotteretal.,1997;Sunderlandetal.,2008).However,Buonoetal.(2009)reportedasignificant

increaseinSWRfollowing4-dofHAtohumidconditions.Thus,STHAappearstobesufficientat

inducing performance-enhancing adaptations (Lorenzo et al., 2010), which may be more

pronouncedafterfluidregulatorystrainfromapermissivedehydrationHAregimen(Garrettetal.,

2014,2011;TaylorandCotter,2006).

63



AdaptationDaysofheatacclimatisation

1 2 3 4 5 6 7 8 9 10 11 12 13 14

HRdecrease ______________

PVexpansion ______________

Tredecrease ______________

RPEdecrease ______________

SweatNaCldecrease __________________

SWRincrease ___________________________

RenalNaCldecrease __________________

Notes:HR,heartrate;NaCl,SodiumChloride;PV,plasmavolume;Tre,rectaltemperature;SWR,sweatrate.

AdaptedfromArmstrongandMaresh,(1991).

Theplateauinresponsesfollowing3-6dHA(table2.9)haspreviouslybeenviewedasfavourable.

However,traditionalmethodsofHAmayconstraintheadaptationandthustheplateaumaysimply

reflectincompleteadaptation.Thereisanabsenceofinformationonthetemporalpatterningof

controlledhyperthermiaHA,thisisproblematicsinceitisessentialthatwherepossibletheduration

ofHAadministeredelicitsoptimaladaptation.

2.17. Heatacclimationdecay

Heatacclimationisatransientprocessandwillgraduallydisappearifnotmaintainedbycontinued

repeated exercise-heat exposure. Literature is consistent in reporting that the physiological

adaptationsassociatedwithHAarerelativelyshort-termandmaydecayonlyafewdaysorweeks

afterremovalfromheatexposure(ArmstrongandMaresh,1991).Thefirstadaptationstooccur

aretypicallythefirsttodecay,withthephysiologicaladaptationsthattakelongertodevelop,such

assudomotorfunction,willhaveaslowerrateofdecay(Taylor,2000).Flourisetal.(2014)assessed

thedecayof14-doffixedintensityHA.TheobservedchangesinTcpersistforatleast2-wksafter

removalfromheatexposure;whilethechangesinHRandHRvariabilitydecayfasterandareonly

64

partlyevidentafter2-wksofnon-exposuretoheat.Furthermore,Garrettetal.(2009)assessedthe

decayof5-dcontrolledhyperthermiaHA following2,9,16and23-dwithoutexposure toheat.

STHAinducedadaptationspermittingincreasedheatloss;theseadaptationspersistedforone,but

not 2-wks following HA. Thus, when using a cross over design to assess two HA methods,

considerationneedstobetakentoensureanadequatewashoutperiodisused.

Furthermore, the re-inductionofHAafter its lossoccursmarkedly faster thanduring the initial

induction period (Tetievsky et al., 2008).Weller et al. (2007) assessed the re-induction of 10-d

controlled hyperthermia HA following 12 and 26-d without exposure to heat. It took 2-4 d

respectivelytorestorethepreviousobservedphysiologicalbenefits.Thus,onceadaptationtothe

heathadbeenattained,thetimethatindividuals’mayspendincoolerconditionsbeforereturning

toahotenvironment,couldbeaslongasonemonthwithouttheneedforextensivere-adaptation

toheat.Thisobservation isconsistentwithresearchconcerningadaptationmemory,withsome

acquiredfunctionalchangesbeingrapidlyrestored(Tetievskyetal.,2008).Thus,whenusingacross

over design to assess two HA methods, consideration needs to be taken to ensure trials are

successfullyrandomisedtoreducetheeffectofHAmemory.

2.18. Females’responsestoheatacclimation

There is a dearthof literature assessing females’ responses toHA. Previously, thephysiological

responses ofmales and females to 10-d fixed intensityHAwere assessedwith females initially

exhibiting lower Tre and HR, despite a lower SWR compared withmales (Avellini et al., 1980).

Following HA, the physiological strainwas similar betweenmales and females, althoughmales

maintained a greater SWR. This study adopted a traditional HA protocol which results in a

progressivedeclineintheadaptationstimulusoverthedurationofHA.Controlledhyperthermia

ensures consistent potentiating stimuli for adaptation throughout the HA period, eliciting

reductionsinthermalstrainandincreasesinworkcapacityduringbothSTHA(Garrettetal.,2012,

2009)andLTHA(Pattersonetal.,2004a),potentiallypromotingmorecompleteadaptation(Taylor

andCotter,2006).Furthermore,Sunderlandetal.(2008)assessedtheresponsesoffemalegames

players responses to STHA. Results suggest, that females fail to establish typical phenotypic

adaptations, namely reduction in Tre and HR despite a 33% increase in intermittent sprint

performance in the heat. These typical adaptive responses have been previously observed in

trainedmalesfollowingSTHA(Buonoetal.,1998;Fujiietal.,2012;Garrettetal.,2011;Poirieret

al., 2015; Racinais et al., 2012); suggesting females may require LTHA to achieve adaptation.

However,duetotheself-pacedexerciseadministeredpreandpostHAbySunderlandetal.(2008),

females were exercising at a higher absolute intensity following HA, suggesting an increase in

65

metabolicheatproduction;potentiallynegatinganyimprovementsinthermoregulationachieved

throughHA.Itremainsunknowntheextenttowhichfemales’adapttothecontrolledhyperthermia

model of HA. The absence of evidence concerning effective STHA protocols in females is

problematicforathletes,sinceSTHAisapreferredregimeasitprovideslessdisruptionfromquality

trainingpriortocompetitioncomparedwithtraditionalHAprotocols.

2.19. Aimofthesis

AHTTusinga runningmodeofexercise,dataon females’phenotypicandcellular responses to

controlledhyperthermiaHAandestablishingoptimisedHAstrategiesforfemalesarerequired.This

thesiswillthereforeconsistofaseriesofexperimentswhichwillfirstlyassesstherepeatabilityand

thesensitivityofaRHTT.Oncethishasbeenestablished,theRHTTwillbeusedtocomparethe

phenotypicandcellularadaptationresponsestocontrolledhyperthermiainmalesandfemales.A

furtherexperimentwill thenbeperformed toevaluateanovelHA strategy in females across a

short-termtimescale.

2.19.1. Researchquestionsarisingfromtheliteraturereview

The literature review has identified a number of areas for investigation still outstanding. The

incidenceofheat-related illnesses in running isapparent,yet there iscurrentlynostandardised

methodtoevaluatearunner’sheattolerance.ThedevelopmentofaRHTTiswarranted.Itiswell

understoodthatcontrolledhyperthermiaHAelicitsmoreoptimaladaptationcomparedwithfixed

intensityprotocols.Aseriesofintegratedphysiologicalandcellularadaptationselicitanenhanced

exerciseheattolerance.Whilstthetemporalpatterningandlengthofexposurerequiredtoelicit

phenotypic and cellular adaptations following controlled hyperthermia HA are becoming well

understoodinmales,littleprogressionhasbeenmadewithregardstofemales’responses.Thus,a

comparisonofmales’andfemales’phenotypicandcellularresponsestocontrolledhyperthermia

HAiswarranted.Oncefemales’temporalpatterningassociatedwithcontrolledhyperthermiaHA

hasbeenidentified,itremainstobedeterminedwhethertherearemoreoptimisedHAmethods

toestablishanacceleratedadaptioninfemales.Passiveheatexposurestosaunasandhotwater

have successfully elicited phenotypic adaptation in males; consideration of these protocols is

requiredinfemales.

66

2.19.2. Proposedresearchstudiesandhypotheses

Thefollowingresearchquestionsandhypothesesareproposedforthisthesis.

Repeatabilityofarunningheattolerancetest

Aim:Toestablishtherepeatabilityofapracticalrunningtesttoevaluateanindividual’sabilityto

tolerateexerciseheatstress.

Hypotheses: Itwasprimarilyhypothesised thatphysiological responsesduring the runningheat

tolerancetestwillhaveastrongpositivecorrelationonrepeatedtrials.Secondly,therewillbeno

differences inphysiological responsesduring the runningheat tolerance testbetween repeated

trials.

Sensitivityofarunningheattolerancetest

Aim: To identify the sensitivity of the runningheat tolerance test to changes in heat tolerance

followingshort-termheatacclimation.

Hypotheses:Itwasprimarilyhypothesisedthattherewillbedifferencesinphysiologicalresponses

duringtherunningheattolerancetestfollowingshort-termheatacclimation.Secondly,therunning

heattolerancetestwillbesensitivetoindividualdifferencesinthemagnitudeofadaptation.

Acomparisonofmaleandfemaletemporalpatterningtoshort-andlong-termheatacclimation

Aim: To determine the sex differences in thermoregulatory, cardiovascular, and sudomotor

adaptationtoshort-termandlong-termheatacclimation.

Hypotheses:ItwasprimarilyhypothesisedthatmaleswillachievegreaterreductionsinTreandHR

followingSTHAcomparedwithfemales.Secondly,thattherewillbenodifferencesinthereduction

inTreandHRbetweenmalesandfemalesfollowingLTHA.Finally,thatfemaleswillachieveagreater

increaseinSWRfollowingSTHAandLTHAcomparedwithmales.

SexcomparisonofleukocyteHsp72mRNAtranscriptionduringheatacclimation

Aim:Toexaminethesexdifferencesinheatshockprotein72mRNAtranscriptionduringcontrolled

hyperthermiaheatacclimationovershort-andlong-termtimescales.

67

Hypothesis:Itwashypothesisedthatheatshockprotein72mRNAresponsewillbelowerinfemales

comparedtomalesacrossthecourseofcontrolledhyperthermiaheatacclimation.

Passiveheatexposureprecedingshort-termheatacclimationacceleratesphenotypicadaptation

infemales.

Aim:Todeterminewhethershort-termheatacclimationprecededbyapassiveheatexposureto

sauna-likeconditionsacceleratedheatadaptationinfemales.

Hypotheses:Itwasprimarilyhypothesisedthatshort-termHAprecededbyapassiveheatexposure

willresultinagreaterplasmavolumeexpansioninfemalescomparedwithheatacclimationalone.

Secondly,short-termheatacclimationprecededbyapassiveheatexposurewillproduceagreater

sudomotoradaptationinfemalescomparedwithheatacclimationalone.

68

3. Generalmethods

________________________________________________________________________________

Thischapterdescribesthegeneralmethodsandmaterialsusedwithintheexperimentalchapters

of this thesis.When additional ormodifiedmeasureswere used, full descriptions are included

withinthemethodssectionoftherelevantexperimentalchapters.

3.1. Ethics,healthandsafety

AllexperimentsreportedwithinthisthesiswereapprovedbytheUniversityofBrightonResearch

Ethics&GovernanceCommitteeandconductedinaccordancewiththeguidelinesoftherevised

DeclarationofHelsinki, 2013.All experimentationwas carriedout in linewith theUniversityof

Brighton’sstandardoperatingproceduresandriskassessmentlaboratoryguidelines.

Experimenters

Therewere at least two experimenters present throughout each experimental trial within this

thesis.Oneexperimenterremainedwithintheenvironmentalchamberattendingtotheparticipant

and one experimenter remained outside the environmental chamber, ensuring the safety of

individualswithin the chamber for the duration of each trial. Furthermore, at least one of the

experimenterspresentduringtheexperimentaltrialswasaqualifiedfirstaider.

Equipmentcleaningprocedure

To avoid contamination, all equipment used was cleaned before and after use. Respiratory

apparatuswassoakedin1%Virkondisinfectant(AntecInternational,UK)foraminimumof10-min

aspermanufacturerinstructions.Apparatuswasthenthoroughlyrinsedanddriedpriortouse.HR

monitorstrapsweresoakedfor10-minin1%Virkondisinfectant,rinsedanddriedfollowinguse.

Allskinthermistorswerecleanedusingalcoholwipes(MölnlyckeHealthcare,Sweden)beforeand

afteruse.All otherequipment including the cycleergometerand themotorised treadmillwere

wipeddownusingdisinfectantsurfacespray(Bioguard,UK)followinguse.

Controlofsubstanceshazardoustohealth

Control of substances hazardous to health formswere completed and passed onto theWelkin

LaboratoriesBiologicalSafetyofficer forapproval foreverypowderandsolutionusedwithinall

69

experimentspresentedwithinthisthesis.Theseformsoutlinedanyknownrisksassociatedwiththe

powderorsolutionandthecorrectstoring,handling,anddisposalprocedures.

Wastedisposal

Rectalthermistorsandallothernon-reusablewasteweredisposedofbyincineration.Allsharps,

suchasneedlesandlancetsforthemeasurementofbloodweredisposedofinadesignatedsharps

bin.Biologicalmaterialandwastewerehandledanddisposedofinlinewithrelevantguidelines.

Criteriaforterminationofexperiments

Writteninformedconsent(Appendix1)andamedicalhistory(Appendix2)wasobtainedfromeach

participantpriortoanytestingandallparticipantswereinformedoftheirrighttowithdrawfrom

thestudyatanytimewithoutobligationtogivereason.ExercisewasterminatedifTre≥39.7°Cin

linewiththeuniversitiesethicallimits,theparticipantwithdrewduetovolitionalexhaustion,the

participantscouldnolongermaintaintheexerciseintensitydespitestrongverbalencouragement,

or the participant demonstrated signs of heat illness including heat syncope, exhaustion,

disorientation, nausea or vomiting. Upon removal from the chamber, appropriate cooling

techniqueswereadopted.Coolingtechniquesinvolvedcoldwateringestion,coldwaterimmersion

of the hands and feet and sitting in front of a fan. Participants were permitted to leave the

laboratorywhenTrereturnedtowithin0.5°Cofbaselinemeasuresobtainedpriortotesting.

3.2. Participants

Recruitment

Participants were recruited via an email to University of Brighton students and through

advertisementonsocialmedia,includingFacebookandTwitter.Thisprovidedbasicdetailsabout

thestudyandcontactdetails fortheprincipalexperimenter.Prospectiveparticipantswerethen

providedwithaverbalexplanationofthestudy,inadditionanexplanationoftherisksandbenefits.

Adetailedparticipantinformationpackwasthenprovided.

Medicalcriteria

Athletes,aged18–35yrs.oldwhoincludedrunningandcyclingwithintheirweeklytrainingand

hadarecenthistoryofcompetinginanenduranceevent,whohadnoknowninjury,respiratory

problems,diabetes,orcardiacproblemswererecruited.Participantswerenotselectedtotakepart

inanyofthestudiesifanycontraindicationswereidentified.Participantsweredeemedsuitableif

70

theyhadbeenabsentfromrepeatedexternalheatexposureforthepreviousthreemonthsandhad

noknownpreviousincidentofheatillness.Participantswereexcludedfromtakingpartiftheywere

takinganydietarysupplementsormedicationotherthanthecontraceptivepill,hadtakenpartin

otherlaboratoryexperimentsinthethreemonthspreceding,hadexperiencedanaphylacticshock

symptomstoneedles,probesorothermedicalequipmentthatmightprecludethemfrominserting

arectalprobe,orhadaknownhistoryofrectalbleeding,analfissures,haemorrhoids,oranyother

conditionoftherectum.Femaleswhohad irregularmenstrualcycleorwhowereusinganother

formofhormonalcontraceptionotherthanthecombinationpillwereexcludedfromtakingpart.

3.3. Facilities

AlltestingtookplaceinBritishAssociationofSportandExerciseSciencesaccreditedlaboratories,

attheWelkinsiteoftheUniversityofBrighton,EastbourneCampus.Allpreliminarytestingtook

placeintheresearchlaboratory.Allexperimentalheattrialswereconductedintheenvironmental

physiology laboratory which housed an environmental chamber 4.5 x 3.5 x 3m high (WatFlow

controlsystem;TISS,Hampshire,UK).

3.4. Pre-trialdietandexercisestandardisation

Experimentaltrialswereconductedatthesametimeofdayforallparticipantstoavoidaneffect

of circadian variation on physiological variables (Atkinson and Reilly, 1996). Experimentation

occurredduring theUKwinter (meanambient temperatureof5°C); therefore,participantshad

beenabsentfromrepeatedexternalheatexposurefortheprevious3months.Participantswere

instructedtorefrainfromhotbaths,saunarooms,steamrooms,andsunbedsduringthethree

months prior to the experimental testing and throughout the testing period. Furthermore,

participantswere instructed to refrain fromexposure tohypobaric or hyperbaric environments

duringthethreemonthspriortotheexperimentaltestingandthroughoutthetestingperiod.

48-hrspriortoconductingthetrials,participantswereinstructedtomaintainnormalhydrationand

diet,andrefrainfromtheconsumptionofalcohol(Yodaetal.,2005),caffeine(DelCosoetal.,2009),

glutamine, generic supplementation and exhaustive exercise. 2-hrs prior to arrival participants

wereinstructedtoslowlyconsume3-5mL.kg-1(250–400mL)ofwatertoensureadequatehydration

(Sawkaetal.,2007).Participantswererecommendedtoconsumeasmallbreakfastconsistingof

30-50 g ofmultigrain cereal with semi-skimmedmilk, or two slices of wholemeal toast with a

toppingofchoice,2-hrspriortoarrival.Participantswereinstructedtomaintainidenticaldietin

theimmediate48-hrspriortoeachexperimentalsession.

71

Hydrationassessment

Toensureequalandadequatehydrationbetweentrialstheassessmentofhydrationstatuswas

performed.When twooutof the following threecriteriawereachieved,adequatehydration to

performthe trialwasassumed;anosmolalityvalueof≤700mOsm.kg-1,aurinespecificgravity

(USG)valueof≤1.020,oraBMwithin1%ofdailyaverage(Sawkaetal.,2007).Theseexperimental

controls were not violated for any participant for any of the preliminary or experimental

procedures.

Urineosmolality

Urineosmolalitywasmeasuredusingahandheldmicroosmometer(AdvancedInstruments Inc.,

Massachusetts,USA).Themicroosmometerwascalibratedprior toeverysampleusingdistilled

water(Osmolality0mOsm.kg-1).Approximately1mLofurinewasplacedintotheosmometerlens,

wherebythesamplewasmeasuredusingwaterfreezingpointdepression.

Urinespecificgravity

Urinespecificgravitywasassessedusingavisualhandheldrefractometer(IndexInstrumentsLtd.,

Cambridge,UK).Therefractometerwascalibratedpriortoeverysampleusingdistilledwater(USG

1.000). Approximately 2 mL of urine was placed onto a glass lens of the refractometer. The

refractometerwasthenhelduptothelight,whiletheexperimenterlookedthroughtheeyelens

andrecordedthevaluesfromthescalewithin.

Repeatability3ofurinaryanalysis

A urine sample was collected and the same sample analysed for osmolality and USG on ten

occasions.Betweenanalysestheosmometerandrefractometerwascalibratedwithdistilledwater.

Technicalerrorasacoefficientofvariation(TE(CV%)),andintra-classcorrelationcoefficientwith

95%confidenceintervals(ICC95%CI)werecalculatedfromduplicatemeasuresof10samples(table

3.1).

3Reliability,repeatability,reproducibilityandagreementaretermsoftenusedinterchangeably.Thetermrepeatabilitywillbeusedconsistentlythroughoutthisthesissinceitspecificallyreferstothevariationinrepeatedmeasurementsmadeonthesameparticipantunderidenticalconditions.

72

Table3.1Repeatabilityofurineosmolalityandurinespecificgravitymeasurement.Mean±SD.

Urineosmolality(mOsm.kg-1) Urinespecificgravity

1 2 1 2

Mean±SD 245±187 245±186 1.016±0.030 1.016±0.030

Difference 0±7 0.000±0.001

TEM 5 0.001

TE(CV%) 2 0.055

ICC(95%CI) 0.99(0.99,1.00);p≤0.001 1.00(0.99,1.00);p≤0.001

Notes:ICC(95%CI),Intra-classcorrelationcoefficientwith95%confidenceintervals;SD,standarddeviation;

TEM,typicalerrorofmeasure;TE(CV%),typicalerrorasacoefficientofvariation.

3.5. Anthropometricassessment

Stature

Staturewasmeasuredusingafixedstadiometer(DetectoPhysiansScales,DetectoScaleCompany,

Missouri,USA).Participantswererequiredtostandverticallyintheanatomicalpositionfacingaway

from the stadiometer scale in the laboratory. The stadiometer armwas lowered until it rested

horizontallyonthemostsuperioraspectofthehead.Thescalewasthenreadtothenearest0.5

cm.

Bodymass

Nude BM was measured using Adam GFK 150 digital body scales (Adam Equipment Inc.,

Connecticut,USA)andrecordedto0.01kg.Thescaleswerecalibratedpriortouse,usinga20kg

weight.MorningBMwasrecordedonfourconsecutiveoccasionspriortotheexperimentaltrials.

Thisprocedurewascarriedoutinaprivateroom,whereparticipantsself-reportedtheirBMtothe

experimenter.

73

Bodysurfacearea

Following measurement of both stature and BM, body surface area (BSA) was subsequently

calculatedusingEquation3.1(DuBoisandDuBois,1916).

Equation3.1Calculationofbodysurfacearea(BSA)

BSA=0.007184xBM0.425xH0.725

Where:BM=bodymassinkg,H=statureinmeters

Skinfolds

Skin fold thickness was determined from the right side of each participant whilst stood in the

anatomicalpositioninambientlaboratoryconditionsof20±1°Cand40±5%RH.Sumofskinfolds

wasdeterminedfromfoursites(DurninandWomersley,1974);thebicep,triceps,subscapular,and

supra-iliacareausingHarpendenskinfoldcallipers(BatyInternational,WestSussex,UK).Skinfold

thicknesswasmeasuredtothenearest2mm.Measuresweretakenintriplicateandtheaverage

recorded.Ifvalueswerenotwithin0.4mmadditionalmeasuresweretaken.

Thebicepskinfoldwaslocatedatthepointontheanteriorsurfaceofthearminthemidlineatthe

levelof themid-acromial-radiale landmark.The tricepskin foldwas locatedat thepointon the

posteriorsurfaceofthearm,inthemidline,atthelevelofthemid-acromial-radialelandmark.The

subscapularskinfoldwaslocated2cmlaterallyandobliquelydownwardsfromtheundermosttip

oftheinferiorangleofthescapula.Thesupra-iliacskinfoldwaslocatedatthepointontheiliac

crest where the midaxilla on the longitudinal axis of the body meets the ilium (Durnin and

Womersley,1974).

Whentheselocationshadbeenmarked,afirmgraspwastakenoftheskinandsubcutaneousfat

ensuringnomuscleorfasciawastakenusingthethumbandindexfinger.Thenthecontactsurface

of thecalliperswasplacedata90-degreeangle to the skin foldapproximately1 cmbelow the

fingers. The pressure was then slightly released between the fingers, ensuring that a greater

pressure was applied by the callipers. The handle of the callipers was then released and the

measurementwasrecordedapproximately4safterthepressurewasreleased.

74

Repeatabilityofskinfoldmeasurement

The skinfold thickness from four sites on eight participants was recorded on two consecutive

mornings.TEM,TE(CV%),andICC95%CIwerecalculatedfromtheseduplicatemeasures(table

3.2).

Table3.2Repeatabilityofskinfoldmeasurements.Mean±SD.

Bicep Tricep Subscapular Supra-iliac

D1(mm) 6.8±2.3 7.0±3.3 10.0±2.5 10.5±2.7

D2(mm) 6.9±2.4 7.1±3.2 10.1±2.5 10.4±2.8

TEM 0.2 0.1 0.1 0.3

TE(CV%) 2.9 1.2 0.7 2.4

ICC(95%CI)0.99(0.98,0.99);

p≤0.001

0.99(0.99,1.00);

p≤0.01

1.00(0.99,1.00);

p≤0.001

0.99(0.98,0.99);

p≤0.001

Notes:ICC(95%CI),Intra-classcorrelationcoefficientwith95%confidenceintervals;SD,standarddeviation;

TEM,typicalerrorofmeasure;TE(CV%),typicalerrorasacoefficientofvariation.

3.6. Menstrualcycle

Timingoftesting

Tocontrolforhormonaleffectsonthermoregulationassociatedwiththemenstrualcycle,female

participantsfilledoutamenstrualcyclequestionnaire(Appendix3)for2monthspriortothetesting

sessionsbeingscheduled.Wherepossible,alltestingwasperformedduringthefollicularphaseof

the menstrual cycle (3-10 d after the onset of menstruation) (Stachenfeld and Taylor, 2014).

However, female participants taking oral contraceptive performed the experimental sessions

duringthenopillorinactivephaseoforalcontraceptiveusewherepossible.

Validityofthemenstrualcyclequestionnaire

Datacollectedinour laboratorydemonstratedthevalidityofmenstrualcyclequestionnairesfor

accuratelydetermining thecorrectphaseof themenstrual cycle.Nineparticipantscompleteda

75

menstrualcyclequestionnairefortwofullcycles.Participantsthenreportedtothelaboratoryon

fiveoccasionsacrosstwofullcycles.ParticipantsreportedtothelaboratoryonD3,D10,andD22

aftertheonsetofmenstruationduringthefirstcycleandthenonD3andD10aftertheonsetof

menstruationduringthesecondcycle.Plasmaconcentrationsof17β-estradiolandprogesterone

werequantifiedtodetermineaccuratelythemenstrualcyclephaseusingcommerciallyavailable

17β-estradiol(ab108667)andProgesterone(ab108670)immunoenzymaticassaykits(Abcamplc,

UK).

Plasmaconcentrationsof17β-estradiolandprogesteroneweresimilaronD3andD10acrosstwo

fullmenstrual cycles (table 3.3). Plasma concentrations of17β-estradiol and progesteronewere

higheronD22inallparticipants(p≤0.05),providingevidencethatamenstrualcyclequestionnaire

isaneffectivemethodtodeterminemenstrualcyclephaseaccurately.

Table3.3Plasmaconcentrationof17β-estradiolandprogesterone.Mean±SD(range).

Day 3 10 22 3 10

17βestradiol

(pg.mL-1)

43±18*

(12-75)

62±30*

(32-127)

211±64

(121-279)

42±23*

(23-89)

43±24*

(24-97)

Progesterone

(ng.mL-1)

1.25±0.63*

(0.90–2.39)

1.10±0.67*

(0.15–1.96)

12.21±16.58

(4.10–50.63)

1.03±0.57*

(0.09–1.68)

1.70±0.68*

(0.05–1.75)

Notes:*denotessignificantdifferencetoD22.

3.7. Environmentalconditions

Ambientlaboratorytemperaturecontrol

Seated rest for themeasurement of resting values and preliminary testing were performed in

ambient laboratory conditions at 20 ± 1°C and 40 ± 5% RH;WBGT = 19°C, using industrial air

conditioning.Barometricpressurewasdeterminedfromaportablebarometer(Weatherstation,

OregonScientific,Oregon,USA).

76

Experimentaltemperatureandhumidity

Allexerciseheatsessionswereperformedinalarge,purpose-builtenvironmentalchamberwith

computer controlledmonitoring andmaintenance of desired environmental conditionswith an

availablerangeof-20°Cto+50°Cand20to95%RH.AsanindexofheatstressWBGTwascalculated.

Unlessotherwisestated,experimentaltrialswereconductedin40°Cand40%RH(WBGT=33°C).

Duringexerciseheat stressexperimental trials,manual recordingof thechamberconditionwas

performedevery5-mintodescribeaccuratelytheenvironmentexperiencedbyparticipants.

3.8. Thermoregulatorymeasures

Rectaltemperature

RectaltemperaturewasmeasuredtoindicateTc,usingageneralpurposedisposableprobe(Henley,

Reading,UK)attachedtoadatameter(MeasSpec4600,measurementSpecialities,VirginiaUSA)

measuredtotheaccuracyof0.01°C.Therectalprobewasmeasuredatadepthof10cmpastthe

analsphincterwithazincoxidetapebungappliedtotheprobepriorto insertiontoensurethe

correctdepthwasattainedandmaintainedthroughouttesting.

Repeatabilityofrectalthermistor

Therepeatabilityoftherectal thermistorwasdeterminedpriortoexercise inarestedstate.An

individualwasexposedtotemperatelaboratoryconditionsinarestedstatefor20-mintostabilise

Tre.MeasurementsofTrewerethentakeneveryminfortwo10-minperiods.TEM,TE(CV%),and

ICC95%CIwerecalculatedfromduplicatemeasures(table3.4).

77

Table3.4Repeatabilityofrectalthermistors.Mean±SD.

RestingTre(°C)

Measures 1 2

Mean±SD 37.4±0.0 37.4±0.02

Difference 0.02±0.02

TEM 0.02

TE(CV%) 0.04

ICC(95%CI) 0.99(0.98,1.00);p≤0.001

Notes: ICC, intra-classcorrelationcoefficient;TEM, typicalerrorofmeasure;TE (CV%), typicalerrorasa

coefficientofvariation;Tre,rectaltemperature.

Skintemperature

Tskwasrecordedusingskinthermistors(EltekLtd,Cambridge,UK)attachedtofoursitesontheright

sideofthebodyconnectedtoaSquirreltemperaturelogger(Squirrel1000series,EltekLtd.,UK).

Thefoursiteswerethechest(midpointofthepectoralismajo),thearm(midpointofthetriceps

brachii lateral head), the upper leg (midpoint of the rectus femoris), and the lower leg

(gastrocnemiuslateralhead)MeanTskwascalculatedusingEquation2.4(Ramanathan,1964).

Repeatabilityoftheskinthermistors

Therepeatabilityoftheskinthermistorwasdeterminedbyrecordingthemeantemperatureoffour

skinthermistorsexposedtoambientconditions.Theambientconditionswerestableat20°Cand

40%RHthroughoutthedatacollection.Theskinthermistorswerelefttostabilisefor20-minand

thenmeasurementswererecordedeveryminfortwo10-minperiods.Datawasthenaveragedover

each10-minperiod.TEM,TE(CV%),andICC95%CIwerecalculatedacrossthetwo10-minperiods

(table3.5).

78

Table3.5Repeatabilityofskinthermistors.Mean±SD.

Meanskinthermistor(°C)

Measures 1 2

Mean±SD 20.3±0.1 20.2±0.1

TEM 0.1

TE(CV%) 0.3

ICC(95%CI) 0.91(0.70,0.98);p≤0.001

Notes:ICC(95%CI),intra-classcorrelationcoefficientwith95%confidenceintervals;SD,standarddeviation;

TEM,typicalerrorofmeasure;TE(CV%)technicalerrorasacoefficientofvariation.

3.9. Cardiopulmonarymeasures

Metabolicgasanalysis

Expired metabolic gas was measured using a breath-by-breath online gas analysis system

(MetalyzerSport,Cortex,Germany).Calibrationwasperformedaspermanufacturer’sinstructions.

Calibration was performed for pressure, volume turbine and gas sensors prior to each use.

Barometric pressure was determined and calibrated to the device from a portable barometer

(GA690,CastleGroupLtd,UK).Volumecalibrationrequiredsimulatedinspirationandexpirationvia

amanualsyringe(3L,HansRudolph,Germany)forfiveacceptablecycleselicitingaflowrateof2

to4L.s-1.According to themanufacturer’s instructions, the flowsensormeasuredvalues to the

accuracyof±2%witharangeof0.05-20L.s-1.Gasconcentrationcalibrationrequiredtwoknown

O2andtwoknownCO2concentrations,knownasgas1andgas2,tobesampleduntilstabilisation

ofsensorshadbeenachieved.Accordingtothemanufacturer’sinstructions,theO2sensorandCO2

sensorsmeasured values to the accuracy of 0.1 Vol%with a range of 0 to 60% and 0 to 13%

respectively.

Inaccordancewithmanufacturer’sguidelines,gas1wasrequiredtorepresentclosetotheinspired

fractionandgas2closetotheexpiredfraction.Gas1wastakendirectlyfromambientconditions

(inspiredfractionO2=0.2093,inspiredfractionCO2=0.0005),Gas2wasdrawnfromacontained

79

gascylinder(expiredfractionO2=0.17,expiredfractionCO2=0.05)intoaclamp-sealedcollection

bag,whichwas immediatelyaffixed to thesample line. All respiratorygasexchangedatawere

averagedovera10speriod.

Heartrate

HeartratewasrecordedusingaPolarHRmonitor(PolarElectroOyo,Kempele,Finland).Achest

strap transmitter was securely affixed to the participant and the sensors dampened to aid

conductivity.ThewristwatchreceiverwasalsoworntoenableliveHRdatarecording.According

tothemanufacturer’sinstructions,theHRmonitorisaccurateduringsteadystateconditionsto±

1%or±1beat.min-1,whicheverislarger.

3.10. Physiologicalstrainindex

Thephysiologicalstrainindex(PSI)wascalculatedfromexercisingTreandHRusingEquation3.2.

Equation3.2Calculationofphysiologicalstrainindex(PSI)

PSI=5(Tret-Treo)*(39.5-Treo)-1+5(HRt-HRo)*(180-HRo)-1

Where:TreoandHRoarethe initialandTretandHRtaresimultaneousmeasurements takenatanytime

(Moranetal.,1998).

3.11. Haematologicalmeasures

Capillarylactatesample

Forcapillarylactatesamplingthefingertipwasfirstcleanedusinganalcoholwipeandlefttoairfor

approximately15s.Theskinwaspuncturedusingasingleuselancet(Acc-ChekSafe-T-Pro,Roche

Diagnosticsltd.,WestSussex,UK).Applyinglightpressuretothesitea~200µlofcapillaryblood

wascollectedfromthefingertipintoaheparin-fluoridecoatedmicrovettetubeandsubsequently

analysed for lactate concentration using an automated analyser (YSI 2300 Stat Plus, YSI UK,

Hampshire, UK). The analyser was calibrated immediately before each session using the

manufacturer’s 5 mmol.L-1 standard, set to self-calibrate every 25-min and verified after each

sessionusingthesamemanufacturerstandard.

80

Repeatabilityofcapillarybloodlactateanalysis

Arestingcapillarybloodsample,combinedwithsamplesatapproximately1mmol.L-1,2mmol.L-1,

3mmol.L-1,4mmol.L-1,and8mmol.L-1wereanalysedinduplicate.TEM,TE(CV%),andICC95%CI

werecalculatedfromduplicatemeasures(table3.6).

Table3.6Repeatabilityofcapillarybloodlactateanalysis.Mean±SD.

Lactateconcentration(mmol.L-1)

1 2

Mean±SD 3.30±2.66 3.32±2.69

TEM 0.03

TE(CV%) 1.02

ICC(95%CI) 1.00(0.99,1.00);p<0.001

Notes:ICC(95%CI),Intra-classcorrelationcoefficientwith95%confidenceintervals;SDstandarddeviation;

TEM,typicalerrorofmeasure;TE(CV%),technicalerrorasacoefficientofvariation.

3.12. Sweatrate

Towel-driednudeBMwasmeasuredandrecordedtothenearestgrammebeforeandimmediately

afteralltrialsasameasureofwholebodySWR.BetweenthesetwomeasuresofnudeBM,fluid

intakewas restricted and valueswere corrected for urine output. Valueswere uncorrected for

respiratoryandmetabolicweightlossesastheywereassumedassimilarbetweentrialsduetothe

matchedexerciseintensityandenvironmentalconditions.SWRwascalculatedandreporteding.hr-

1usingEquation3.3.SWRwasalsocalculatedandreportedrelativetoBSA(SWRBSA) ing.hr-1.m2

usingEquation3.4.

Equation3.3Calculationofwholebodysweatrate(SWR)

SWR(g.hr-1)=[BMpre(g)–(BMpost(g)+urineoutput(g)]/exerciseduration(min)*60

Where:BMpre=bodymasspriortobeginningthetrial,BMpost=bodymassfollowingthetrial.

81

Equation3.4Calculationofwholebodysweatraterelativetobodysurfacearea(SWRBSA)

SWRBSA(g.hr-1.m2)=SWR(g.hr-1)/BSA(m2)

Where:BSA,bodysurfacearea;SWR,sweatrate.

3.13. Perceptualscales

Ratingofperceivedexertion

The rating of perceived exertion scale (RPE), also known as the Borg scale (Borg, 1962), is a

subjectivetooltoassesssubjectiveperceptionofeffortduringexercise.RPErangedfrom6(very,

verylight),through13(somewhathard),to20(very,veryhard)alonga15-pointscale(figure3.1).

RPEhasbeenreportedtobecloselyrelatedtobothmetabolicandcardiac intensityparameters

(Scherr et al., 2013). Participants were provided with standardised instructions that provided

specificcommandsonhowtoreportoverallfeelingsofexertionthroughouttheexperimentaltrials.

Thestandardisedinstructionsincludedclearunderstandingofanchoringthetopandbottomratings

topreviousexperiencesofnoexertionatall(RPE=6)andmaximalexertion(RPE=20)(Maugeret

al.,2013).Withinthisthesis,RPEwasrecordedat10-minintervalsthroughouttheRHTTandHA

sessions.DuringthepreliminarylactatethresholdandVgO2peaktest,RPEwasrecordedinthefinal

15sofeachstage.

Thermalsensation

The thermal sensation (TS) scale (Toner et al., 1986) is a subjective tool to assess thermal

perception.TSranges from0.0 (unbearablycold), through4.0 (comfortable), to8.0 (unbearably

hot)alonga17-pointscale(figure3.1).TShasbeenreportedtobecloselyrelatedtomeasuresof

ambient air temperature,metabolic rate, Tre, and Tsk during exercise (Gagge et al., 1969). The

standardisedinstructionsprovidedtotheparticipantsincludedclearunderstandingofanchoring

the top and bottom ratings to previous experiences of being unbearably cold (TS = 0) and

unbearablyhot(TS=8).WithinthisthesisTSwasrecordedat10-minintervalsthroughouttheRHTT

andHAsessions.

82

Figure3.1Ratingofperceivedexertion(RPE)andthermalsensation(TS)scales.

3.14. Cyclingexercisetrials

Cycleergometry

All cycle ergometry was performed on a cycle ergometer (Monark 874E, Monark Exercise AB,

Vansbro,Sweden).Foralltrialstheseatheightwassettoensurea25°angleinthekneeflexion

whenat6o’clockpedalcrankpositionwasattained.Powerwascontrolledbymanipulatingthe

resistanceonthesuspendedweightpanusinga fixedcadenceof80revolutionspermin (rpm).

Calculationsofpoweroutputsinwatts(W)duringcyclingergometryweremadeasfollows.

Equation3.5Calculationofworkduringcycleergometry

Work(J)=Force(N)xDistance(m)

=Massontheweightpan(kg)xAccelerationduetogravity(m.s-1)xPedalrevolutions(r.s-

1)xFlywheeldistanceperrevolution(m)

83

Equation3.6Calculationofpowerfromworkduringcycleergometry

Power(W)=Work(J)/Time(s)

Cyclingpeakoxygenuptake

Agradedexercisetestwasperformedusingacycleergometerintemperatelaboratoryconditions

(WBGT=19°C)todetermineparticipants’VgO2peak.Participantsperformeda5-minwarmupat60

Wandwereinformedtomaintainaconstantcadenceof80rpm.Thecyclingintensitywassetto

80Wand resistancewasapplied to the flywheel toelicita16 to24W.min-1 increase (selected

depending on the BM of the participant). These intensities were selected to ensure volitional

exhaustionwas reached in8-15min. In the final10 sofeachstageHRwas recorded.VgO2,and

respiratoryexchangeratio(RER)weremeasuredcontinuouslyandaveragedforeach10speriod

throughouteachstage.Thetestwasterminatedwhenparticipantsreachedvolitionalexhaustion

and/or the cadence could no longer be maintained at 80 ± 5 rpm despite strong verbal

encouragement.3-minfollowingthecompletionofthetestacapillarybloodsamplewascollected

fortheanalysisoflactateconcentration.VgO2peakwasidentifiedatthepowerassociatedwiththe

highestVgO2averagedover10sec.VgO2peakwasdeterminedwhenthreeoutofthefollowingfour

criteriawhereachieved;aplateauinVgO2(increasebylessthan2mL.kg-1.min-1),anRER≥1.15,HR

reached 10 beats.min-1 from age predicted max and blood lactate ≥ 8 mmol.L-1. The protocol

adoptedwasaccordingtotheBritishAssociationofSportandExerciseScienceGuidelines(Davison

andWooles,2007).

Heatacclimationprotocol

ThedailyHAsessionsconsistedofa90-minexposure to40°C,40%RH (WBGT=33°C).Exercise

intensitywassetatcycling65%VgO2peak(Appendix4)fromtheoutsetandadjustedwithwork:

restintervalstoachieveandmaintainaTre~38.5°C(Garrettetal.,2012;Pattersonetal.,2004a),or

if participants were unable to maintain a cadence of 80 ± 5 rpm despite strong verbal

encouragementAn example of this protocol for one participant is presented in Figure 3.2. The

controlledhyperthermiamodelofHAwasselectedsinceitensuresaconstantadaptationstimulus

(Foxetal., 1963a), and is reported to result inperformanceandphysiological improvements in

highlytrainedathletes(Garrettetal.,2012).Acyclingmodeofexercisewasselectedsincethereis

lessvariabilityinefficiencycomparedwithrunningbetweenparticipantsthus,ensuringasimilar

stress.Furthermore,duetotheconsecutivenatureoftheHAsessions;cyclingincurslessmuscle

84

damage comparedwith running, subsequently reducing the chance of participants incurring an

injury.

Figure3.2.Exampleofrelativeexerciseintensityelicitedduringa90-mincontrolledhyperthermia

heatacclimationsession.

Heatacclimationintensitypilotstudy

ApilotstudywascarriedouttodeterminetheintensityrequiredtoelicittherequiredriseinTre.

TheintensityneededtobehighenoughtoincreaseTcrapidlyyetlowenoughthatparticipantswere

abletosustaintheintensityandnotincurtoomuchfatigueduetotheconsecutivedaysoftraining.

A25-yr.oldphysicallyactivefemale(stature,1.78m;BM,70kg,VgO2peak,3.61L.min-1at208W)

cycledonthreeoccasionsat55%,65%,and75%VgO2peak.Tre,HR,RPE,andTSwererecordedat

5-min intervals throughout the three trials. Exercise was terminated when Tre reached 38.5°C.

DurationforTretoreach38.5°C,therateofTreincrease(Equation3.7),meanHR,andpeakRPEand

TSwererecordedintable3.7.

Equation3.7CalculationofrateofTreincrease

RateofTreincrease=(∆Tre(°C)/timetaken(min))*60

Where:∆Tre=totalchangeinrectaltemperature

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Exerciseintensity

(%ofV

O2pe

ak)

Time(min)

85

Theresultssuggestedthat65%VgO2peakwasofasufficientintensitytoelicittherequiredincrease

in Tre in 30-min. These findings are similar to the work of Périard et al. (2012) who reported

physiologicalresponsesandtimetoexhaustionwhencyclingat60%and75%VgO2peak.Duringthe

60%VgO2peak trialparticipants terminatedexerciseat~59-min,havingexperiencedaTre riseof

2.1°C,HR~96%maxHR,RPEof18andbloodlactate4.8±2.5mmol.L-1.However,duringthe75%

VgO2peaktrialparticipantsterminatedexerciseafter~27-minhavingexperiencedaTreriseof4.2°C,

HRat ~99%HRmax,RPEof 20 andblood lactate10.9±4.8mmol.L-1.Acknowledgmentof this

publisheddatacombinedwiththepilotworkpresentedsuggeststhataninitialstartingintensityof

65%VgO2peakwouldbesufficient toelicitan increase inTc tothedesired limitof38.5°Cwithin

approximately30-min;providinga60-minstimuluswithTre≥38.5°C.

Table3.7PhysiologicalandsubjectiveresponsetocyclingatsetpercentagesofVgO2peak.

55%VcO2peak 65%VcO2peak 75%VcO2peak

DurationtoTre38.5°C(min) 35 30 25

RateofTreincrease(°C.hr-1) 2.43 3.02 3.29

MeanHR 162 167 170

RPEat38.5°C 14 16 17

TSat38.5°C 6.5 6.5 6.5

Notes:HR,heartrate;RPE,ratingofperceivedexertion;Tre,rectaltemperature;TS,thermalsensation

3.15. Runningexercisetrials

Treadmillrunning

All running based trials were performed on a motorized treadmill (PPS55 sport-1, Woodway,

Germany).Participantswerefamiliarisedwithhowtoreducethespeedorstopthetreadmillifthey

wishedtoterminatethetrialearly.Otherwise,thespeedwascontrolledsolelybytheexperimenter

duringalltreadmilltrials.

86

Runninglactatethreshold

A graded exercise test was performed using a motorized treadmill in temperate laboratory

conditions(WBGT=19°C)todetermineparticipants’lactatethreshold.Participantsperformeda5-

minwarmupataspeed1km.hr-1belowthestartingspeed;thiswastypicallyaverylightjog(7

km.hr-1to9km.hr-1).Theinitialrunningspeedforthetestwassetbetween8km.hr-1and10km.hr-

1with a 1% gradient (Jones andDoust, 1996). Participants then performed five to nine, 3-min,

incremental (0.8 km.hr-1) stages on a treadmill. VgO2 and RER were recorded continuously

throughoutthestageandaveragedforeach10speriod.Inthefinal10sofeachstageHRandRPE

wererecorded.Oncompletionofeachstage,exercisewasmomentarilypausedandlactatesample

wastaken.Thetestwasterminatedwhenlactatereached4.0±0.5mmol.L-1.Lactatethresholdwas

determinedusingthepointatwhichbloodlactateincreased1mmol.L-1aboverestingvalue(Jones

and Doust, 1998). The protocol adoptedwas according to the British Association of Sport and

ExerciseScienceGuidelines(Jones,2007).

Runningpeakoxygenuptake

Followinga15-minrecoveryfromdeterminationofthelactatethreshold,participantsperformed

1-minincremental(1%gradient)stages.OnthecompletionofeachstageVgO2,RERandHRwere

recorded. The testwas terminatedwhen participants reached volitional exhaustion and/or the

speed of the treadmill could no longer be maintained despite strong verbal encouragement.

Immediatelypost and3-min following the completionof the test, a capillaryblood samplewas

collected for the analysis of lactate concentration. VgO2 peak was identified at the intensity

associatedwiththehighestVgO2averagedover10s.VgO2peakwasdeterminedwhenthreeoutof

thefollowingfourcriteriawhereachieved;aplateauinVgO2(increaseof≤2mL.kg-1.min-1),anRER

≥1.15,HRreached10beats.min-1fromagepredictedmax,andbloodlactate≥8mmol.L-1.

3.16. Heattolerancetests

Runningheattolerancetest

TheRHTTwasperformedonamotorizedtreadmillandconsistedof30-minrunningat9km.hr-1

and2%gradient inambientconditionsof40°Cand40%RH(WBGT=33°C).Following20-minof

seatedrest intemperatureconditions,Tre,HR,Tsk,RPE,andTSwererecorded.Participantsthen

enteredtheenvironmentalchambertoperformtheRHTT;whereTre,HR,andTskwererecordedat

5-min intervals and RPE and TS at 10-min intervals. Towel-dried nude BM was measured and

recordedtothenearestgrammebeforeandafteralltrialsasameasureofSWR.

87

Runningheattolerancetestpilotwork

Introduction

TheIsraeliDefenceForce(IDF)developedawalkingHTTtoevaluateifmilitarypersonnel,whohad

experiencedexertionalheatillness,weresafetoreturntoduty.Theprotocolconsistsof120-min

walkingonatreadmillatapaceof5km.hr-1anda2%gradientinambientconditionsof40°Cand

40%RH(WBGT=33°C).At theendof theexposure,heattolerancewasdefinedasapeakTre≤

38.0°C,peakHR≤120beats.min-1,andSWR≥780g.h-1.Heatintolerancewasdefinedas,peakTre

>38.5°C,peakHR>145beats.min-1,(Moranetal.,2007)andapeakPSI≥6(Moranetal.,2004).

There is an instant elevation in the rate of thermogenesis at the onset of physical activity. As

exerciseintensityincreases,especiallyunderhotenvironmentalconditions,athermalimbalance

persistsresultinginacontinuallypositiverateofchangeinbodyheatstorage,increasingbodyheat

contentandasustainedriseinTcgivingagradedincreaseofheatstrain(Jay&Kenny,2007).There

areseverallimitationsassociatedwiththeIDFHTT,whicharemoreapparentwhenconductingthe

testonenduranceathletes(Johnsonetal.,2013).Endurancerunnerstypicallyrunathighintensities

for shorter durations than military personnel. The intensity of the protocol of the IDF HTT is

considerablybelowwhatanathletewouldtrainandcompete.Thus,theIDFHTTmisrepresentsthe

metabolicheatproductionenduranceathletesmayexperienceandpotentiallymaymisdiagnose

theirsusceptibilitytoahyperthermicstate.

Theaimofthispilotstudywastodeterminewhethertherewasasimilarpatternofheattolerance

andheatintolerancebetweentheIDFHTTandtheRHTT.ItwashypothesizedthattheRHTTwould

have a strong positive relationship for the participants’ thermoregulatory and cardiovascular

measuresduringtheIDFHTTandtheRHTT.

Materialsandmethods

Sixteen (8 males; 8 females) healthy individuals volunteered and provided written informed

consenttoparticipateinthepilotstudy(mean±standarddeviation(SD),age,23±5yrs.;BM,67.07

±10.96kg;stature,1.76±0.01m).

Participantsperformedtwotrialsinarandomisedorderseparatedby5-7d.TheIDFHTTinvolved

120-minwalkingat5km.hr-1and2%gradientinhotambientconditionsof40°Cand40%RH(WBGT

= 33°C). The RHTT was conducted using the previously described procedure. Nude BM was

88

measuredpre-andpost-trialasameasureofSWR.At5-minintervalsthroughoutthetrials,HRand

Trewererecorded.

AlldatawerefirstcheckedfornormalityusingtheShapiro-Wilkmethod.Asameasureofretest

correlation,ICCwith95%CIwerecalculatedforeachphysiologicalmeasureduringIDFHTTandthe

RHTT.Pairedsampledttestswerecalculatedtoidentifyanydifferencesinphysiologicalmeasures

betweentheIDFHTTandtheRHTT.Alldatawasanalysedusingastandardstatisticalpackage(SPSS

version20.0),andreportedasmean±SD.Statisticalsignificancewasacceptedatthelevelofp≤

0.05.

Results

Table3.8presentsthemean±SDdataforthephysiologicalvariablesmeasuredduringtheIDFHTT

andtheRHTT.TherewerenoobserveddifferencesbetweentheIDFHTTandtheRHTTinmeasures

ofTre rest (t (15)= -0.597,p=0.559)andHRrest (t (15)=1.370,p=0.191).However, therewere

significantdifferencesbetweentheIDFHTTandtheRHTTinmeasuresofpeakTre(t(15)=-5.567,p

≤0.001),peakHR,(t(15)=-22.753,p≤0.001)andSWR(t(15)=-8.180,p≤0.001).

Table3.8PhysiologyvariablesmeasuredduringtheIDFHTTandtheRHTT.Mean±SD.

PhysiologicalVariables IDFHTT RHTT

Trerest(°C) 37.17±0.26 37.18±0.26

Trepeak(°C) 38.30±0.28 38.82±0.47*

HRrest(beats.min-1) 65±8 64±8

HRpeak(beats.min-1) 135±18 182±15*

SWR(g.hr-1) 637±188 1721±675*

Notes:HR,heartrate;IDFHTT,IsraeliDefenceForceheattolerancetest;RHTT,runningheattolerancetest;

Tre,rectaltemperature;andSWR,sweatrate.*denotessignificantlydifferencetoIDFHTT(p≤0.05).

Figure3.3presentstherawdataforTrerestandHRrest.Strongcorrelations(ICC(95%CI))were

observedbetweentheIDFHTTandtheRHTTforTrerest(r=0.938(0.822-0.978),p≤0.001)andHRrest

(r)=0.959(0.881-0.986),p≤0.001).Figure3.4presentstherawdataforTrepeakandHRpeak.Strong

correlations(ICC(95%CI))wereobservedbetweentheIDFHTTandtheRHTTforTrepeak(r=0.702

89

(0.147-0.896),p=0.012)HRpeak(r=0.932(0.805-0.976),p≤0.001).Furthermore,therewasamedium

correlation(ICC(95%CI))observedbetweentheIDFHTTandtheRHTTforSWR(r=0.599(0.147-0.860),

p=0.043).

Figure3.3Restingrectaltemperature(Tre)andrestingheartrate(HR)duringtheIsraeliDefence

Forceheattolerancetest(IDFHTT)(x-axis)andrunningheattolerancetest(RHTT)(y-axis)formales


8F).

40

50

60

70

80

90

100

40 50 60 70 80 90 100HR

rest(R

HTT)

HRrest(IDFHTT)

MalesFemales

36.4

36.8

37.2

37.6

38.0

36.4 36.8 37.2 37.6 38.0

T rerest(R

HTT)

Tre rest(IDFHTT)

MalesFemales

90

Figure3.4Peakrectaltemperature(Tre)andpeakheartrate(HR)duringtheIsraeliDefenceForce

heat tolerance test (IDF HTT) (x-axis) and running heat tolerance test (RHTT) (y-axis) formales


8F).

Conclusion

TheRHTTwascomparedagainsttheestablishedIDFHTT,sinceithasbeenusedtodifferentiate

betweenheattolerantandheatintolerantmilitarypersonal(Moranetal.,2007).Theaimofthis

pilotstudywastodeterminewhetherthephysiologicalresponsesduringtheIDFRHTTcorrelated

withthephysiologicalresponsesduringtheRHTT.ClassicmarkersofheattoleranceandHA,namely

Trerest,Trepeak,HRrest,HRpeak,andSWRwereusedforanalysis.Themainfindingfromthispilot

studywasthatthereisastrongpositivecorrelationbetweentheIDFHTTandtheRHTT(ICC>0.59).

Inaddition,datapresentedsuggestthattheRHTTiscapableofdifferentiatingbetweenindividual’s

heat tolerancewith individual’s responsessittingalongacontinuum, (figure3.2and figure3.3);

providingevidencethata30-minHTTadoptingarunningmodeofexerciseisofasufficientduration

andintensitytoestablishdifferencesinheattolerancebetweenindividuals.Furthermore,theRHTT

isatimeefficientalternativetotheIDFHTTthatoffersspecificitytoendurancerunners.

37.6

38.0

38.4

38.8

39.2

39.6

40.0

37.6 38.0 38.4 38.8 39.2 39.6 40.0

T repe

ak(R

HTT)

Tre peak(IDFHTT)

Males

Females

90

110

130

150

170

190

210

90 110 130 150 170 190 210

HRpeak(RHT

T)

HRpeak(IDFHTT)

Males

Females

91

3.17. Statisticalanalysis

3.17.1. Poweranalysis

ThenumberofparticipantsrequiredforeachexperimentalchapterwasdeterminedusingG*Power

version3.1inaccordancewithestablishedguidelinesforaprioridetermination(Farrokhyaretal.,

2013)Thevalueofαandβweresetas0.05and0.8,respectively.

3.17.2. Normalityandsphericityofdata

Normaldistribution

Datawascheckedforskewnessandkurtosistodeterminewhetherthedistributionofscoreswas

approximatelynormal.Ifvaluesofskewnessandkurtosiswerebetween-1.96and1.96datawas

assumednormallydistributed(Field,2013).

Furthermore, the Shapiro-Wilk method was used to compare the scores in the data set to a

normallydistributedsetofdatawithanidenticalmeanandSD.Ifthetestwasnon-significant(i.e.

p≥0.05),thenthedistributionwasdeemednotsignificantlydifferentfromthenormaldistribution

anddatawasassumednormallydistributed(Field,2013).

Sphericity

SphericitywasassessedusingtheMauchly’stest,whichteststhehypothesisthatthevarianceof

thedifferencesbetweenconditionsisequal.IfMauchly’steststatisticwassignificant(p≤0.05),it

wasconcludedthat thereweresignificantdifferencesbetweenthevariancesofdifferencesand

therefore,theconditionofsphericitywasnotmet.Iftheassumptionofsphericitywasnotmetthe

degreesoffreedomwereadjustedusingtheGreenhouse-Geissermethod.IfMauchly’steststatistic

wasnon-significant(p≥0.05)thenitwasconcludedthatthevarianceofdifferenceswereabout

equal(Field,2013).

3.17.3. Repeatabilitystatistics

Typicalerrorofmeasure(TEM)

WithinthisthesistheTEMwascalculatedtorepresenttheabsoluterepeatabilityofmeasurements

used.TEMwascalculatedfromtheSDofthedifferencescorebythesquarerootoftwo(Hopkins,

2000a).

92

Equation3.8Calculationoftypicalerrorofthemeasure(TEM)

TEM=SDdiff/√2.

Where:SDdiff=standarddeviationofthedifferencebetweentherepeatedmeasures;√2=square

rootof2.

Coefficientofvariation

WithinthisthesistheTEMhasbeenexpressedasapercentageofitsrespectivemeantoformthe

technicalerrorasacoefficientofvariation(TE(CV%))(Hopkins,2000a).

Equation3.9Calculationoftechnicalerrorasacoefficientofvariation(TE(CV%))

TE(CV%)=(TEM/Mean)*100

Where:TEM=typicalerrorofmeasure,Mean=themeanofthemeanofthepairedsamples

Intraclasscorrelationcoefficient

WithinthisthesisICCwith95%CIwerecalculatedusingSPSSasameasureofretestcorrelation.

Priortoconductingthistestdatawasfirstcheckedfornormalityaspreviouslydescribed.

Equation3.10Calculationofintraclasscorrelation

ICC=(SD2–sd2)/SD2

Where:SD=betweensubjectstandarddeviation,sd=withinsubjectstandarddeviation

3.17.4. Statisticaltestsofdifference

Statisticalsignificancelevel

Withinthisthesisastatisticalsignificancelevelwassettodescribetheprobability(p)ofseeinga

differenceasbig(orbigger)thantheoneyousaw,giventhatthereisnodifference.Thethreshold

93

pvalueforallexperimentswithinthisthesiswassetat5%thus,giving95%confidencethatthere

wasadifferenceorassociation.Withinthisthesis,apvalue≤0.05indicatesthattheobservedeffect

isunlikelytohavearisenpurelybychance,providingevidencetorejectthenullhypothesis.

TypeIandtypeIIerror

Astatisticalsignificancelevelof0.05wasselectedwithinthisthesistoavoidthechancesoftypeI

and type IIerror.Type Ierroroccurswhenyou incorrectly reject thenullhypothesis.Thus,you

wouldsuggestarelationshipordifference,wheninrealitynoneexists.Thistypicallyoccurswhen

thestatisticalsignificancelevelistoohigh(e.g.p≤0.1).Incontrast,typeIIerroroccurswhenyou

accept the null hypothesis when it is false. Thus, you would suggest no relationship, when a

relationshipexists.Thistypicallyoccurswhenthestatisticalsignificancelevel istoolow(e.g.p≤

0.01).

Pairedsamplesttest

Withinthisthesispairedsamplettestswereusedtodeterminestatisticalsignificancebetweentwo

experimentalconditions,whenthesameparticipantswereusedduringbothconditions.Theratio

ofthesevariancesisknownasthet-ratio.Priortoconductingthettest,datawerefirstcheckedfor

skewnessandkurtosistodeterminewhether

Analysisofvariance(ANOVA)

Analysisofvariance(ANOVA)isawayofcomparingtheratioofsystematicvariancetounsystematic

varianceinanexperimentalstudy.TheratioofthesevariancesisknownastheF-ratio.ANOVAisa

collection of statistical models used to analyse the difference among group means and their

associatedprocedures.PriortoconductinganANOVAwithintherespectivestudiesdatawasfirst

checkedfortheassumptionofnormaldistributionandsphericityaspreviouslydescribed(3.17.2.).

3.17.5. Effectsizes

Withinthisthesis,effectsizeshavebeenusedtopresentthemagnitudeofthereportedeffectsin

a standardised metric, providing information on the practical significance of the findings. The

selectedeffect size reported for each statistical test,wasbasedon the recommendations from

Lakens'(2013)reviewoncalculatingandreportingeffectsizes.

94

Cohen’sd

Inthisthesis,effectsizesforttestshavebeenreportedasCohen’sd.Cohen’sdhasbeenusedto

describethestandardisedmeandifferenceofaneffect.ACohen’sdvalueof0.2referstoasmall

effect,0.5asamediumeffectand0.8asalargeeffectbasedonbenchmarkssuggestedby(Cohen,

1988).Cohen’sdinbetweensubjects’designcanbereadilyinterpretedasapercentageoftheSD

suchthataCohen’sdof0.5meansthedifferenceequalstohalfaSD.

Equation3.11CalculationofCohen’sd(d)

d=(X1–X2)/SD

Where:X1=meanfirstsetofdata,X2=meansecondsetofdata,SD=standarddeviation

Partialetasquared

In this thesis, effect sizes forANOVAanalyses, havebeen reported as partial eta squared (np2)

(LevineandHullett,2002).ThiswascalculatedbySPSS.Partialetasquaredexpressesthesumof

squaresoftheeffectinrelationtothesumofsquaresoftheeffectandthesumofsquaresofthe

errorassociatedwiththeeffect.

3.17.6. Posthocanalysis

Tocombatthefamilywiseerrorrateassociatedwithmultiplecomparisons,thelevelofsignificance

forindividualtestswasalteredusingBonferronicorrections.TheBonferronimethodswasselected

withinthisthesisasitisextremelyrobust(althoughslightlyconservative)andcontrolsthealpha

levelregardlessofthemanipulation.IndoingthisitensuresthatthecumulativetypeIerrorremains

below0.05(Field,2013).

Anyadditionalstatisticalanalysesusedwiththisthesisareoutlineintherespectivechapter.

95

4. Repeatabilityofarunningheattolerancetest

________________________________________________________________________________

4.1. Abstract

AtpresentthereisnostandardisedHTTprocedurethatusesrunningasamodeofexercise.The

current study aimed to establish the repeatability of a practical running test to evaluate an

individual’s ability to tolerate exercise heat stress. Sixteen (8M, 8F) participants performed the

RHTT(30-min,9km.hr-1,2%elevation)ontwoseparateoccasionsinahotenvironment(40°Cand

40%RH).TherewerenodifferencesinpeakTre(RHTT1:38.82±0.47°C,RHTT2:38.86±0.49°C,r=

0.93,TEM=0.13°C),peakTsk(RHTT1:38.12±0.45°C,RHTT2:38.11±0.45°C,r=0.79,TEM=0.30°C),

peakHR(RHTT1:182±15beats.min-1,RHTT2:183±15beats.min-1,r=0.99,TEM=2beats.min-1),

orSWR(RHTT1:1,721±675g.hr-1,RHTT2:1,716±745g.hr-1,r=0.95,TEM=162g.hr-1)between

RHTT1andRHTT2(p≥0.05).Resultsdemonstrategoodagreement,strongcorrelationsandsmall

differencesbetweenrepeatedtrials,andtheTEMvaluessuggestlowwithin-participantvariability.

TheRHTTwaseffectiveindifferentiatingbetweenindividuals’physiologicalresponses;supporting

aheattolerancecontinuum.ThefindingssuggesttheRHTTisarepeatablemeasureofphysiological

strainintheheatandmaybeusedtoassesstheeffectivenessofacuteandchronicheat-alleviating

procedures,whenusingarepeatedmeasuresdesign.

4.2. Introduction

Duringexercise inahotenvironment,activemusclesperformworkcausingan increase inbody

heatcontent.Thesechangesaremodulatedbytherateofrelativeheatproduction(Cramerand

Jay,2014),andrepresenttherateofchangeinbodyheatstorage,whichinturnreflectsthebalance

betweenmetabolicheatproduction,heatabsorbedfromtheenvironmentandtotalbodyheatloss

(Jay and Kenny, 2007). Individuals vary in their ability to withstand heat stress, with some

demonstratingadecreasedcapabilitytodissipateheatandgreaterriseinbodyheatcontentunder

the same exercise heat stress (Epstein, 1990). These individuals have been described as heat

intolerant and are often characterized by an earlier and greater rise in Tc, a greater storage of

metabolicheat,ahigherphysiologicalstraintomoderateintensityexerciseintheheat,andreduced

sweatingsensitivity(Epsteinetal.,1983;Moranetal.,2004).

Anindividual’sheatintolerantstatemaybetemporaryorpermanent(Epstein,1990;Moranetal.,

2007;Ruelletal.,2014),stemmingfromtransientpredisposingfactors,suchasanacuteinjuryto

96

thethermoregulatorycentre,insufficientHA,dehydrationorinfectiousdisease(Epstein,1990).In

addition,alastingthermoregulatorydysfunctionmaystemfromconditionssuchascardiacdisease,

impairmenttosweatglands(Epstein,1990),ordifferencesingeneexpression(Moranetal.,2006).

Congenital factors such as ectodermal dysplasiamay also compromise heat tolerance in some

individuals(Epstein,1990).Asidefromthesepredisposingfactors,thehighexerciseintensitythat

endurancerunnersexperienceduringcompetitionscombinedwithextremeambientconditions,

mayelicitunavoidableuncompensableheatproduction.Theevaporativeheatlossrequirementto

maintainathermalsteadystateexceedsthemaximalevaporativecapacityoftheindividualinthe

given environment causing a continual rise in Tc. Thework by Nielsen (1996) provides data to

suggestamarathonrunnermayexperienceuptoa1.0°CriseinTcevery~9-minwhenracinginhigh

ambientconditions(≥35°C,≥60%RH),whenradiantandconvectiveheatlossisnegligible.This

rate of rise in Tcwould result in the runner reaching a Tc of 40.0°Cwithin 25-30min,with the

immediatedangersofheatexhaustion.Highincidenceofexertionalheatillnesshasbeenreported

inlongdistancerunners,with31%and53%ofthetotalnumberofcasesofexertionalheatillness

during the 1992 New Orleans U.S. Olympic Trials and the 1996 Atlanta Olympics, respectively

(Martin,1997).Whetherheatintoleranceispermanentoracquired,theconsequencesofexertional

heatillnessamongenduranceathletesemphasisesthevalueofatesttoevaluateanindividual’s

abilitytowithstandexerciseheatstress.

ExperimentalprocedureshavebeenusedtocauseariseinTcunderrestingandexerciseconditions

tochallengethethermoregulatoryresponses(Inoueetal.,2005;Johnsonetal.,2013;Kenneyand

Hodgson,1987;Montainetal.,1994)andthusassesstheabilityofanindividualtowithstandheat

stressandtoevaluateheatdissipatingmechanisms.TheIsraeliDefenceForce(IDF)developeda

HTTtoevaluatewhetherexertionalheatillnessinmilitarypersonnelwastemporaryorpermanent,

supportingasafereturntoduty(Moranetal.,2004).Theprotocolinvolves120-minwalkingona

treadmillatapaceof5km.hr-1anda2%gradientinhotambientconditionsof40°Cand40%RH

(WBGT=33°C).Heattoleranceisdeterminedattheendoftheexposure,usingcriteriaofpeakTre

≤38.0°C,peakHR≤120beats.min-1,andSWR≥780g.h-1.Deviationsfromthespecifiedcriteria

indicateagreaterstateofheatintolerance,whereasapronouncedplateauinbothTreandHRisa

definitivesignofheattolerance.

97

There is an instant elevation in the rate of thermogenesis at the onset of physical activity. As

exercise intensity increases,especially inanuncompensable4environment,a thermal imbalance

persists.Thisresultsinacontinuallypositiverateofchangeinbodyheatstorage,increasingbody

heatcontentandasustainedriseinTc,givingagradedincreaseofheatstrain(JayandKenny,2007).

TheIDFHTTmaybeappropriateforspecificoccupationalsituationsduetothelow-to-moderate

intensitycoupledwiththelongexposuretimethatislikelytobeexperiencedinmilitaryscenarios.

AcknowledgingtheworkcarriedoutbytheIDF,limitationsassociatedwiththeHTTremainwhen

examiningendurancerunners.Exertionalheatillnessiscompoundedbyuncompensableheatstress

whichisinturn,influencedbythedurationandintensityofexercise.Therelativeworkintensityof

an endurance runner training and competing in the heat is markedly higher compared with

occupationalactivities.Therefore,theIDFHTTmaynotbeapplicabletoanendurancepopulation

duetothedurationandintensityoftheprotocol.ThelowintensitynatureofthecurrentHTTwill

not reflect themetabolic heat production endurance runners experience andmaymisdiagnose

theirsusceptibilitytoahyperthermicstate,pointingtothebenefitofaRHTT.Atpresentthereisno

standardised HTT procedure adopting a runningmode of exercise and such a test would offer

greaterecologicalvalidityforendurancerunners.

Moran and colleagues (2004) assessed the heat tolerance of nineteen male participants and

concludedthatthedurationofaHTTcannotbeshorterthan120-min,sincetoleranceat60-min

wasunabletopredicttoleranceat120-min.TheworkbyEpsteinandcolleagues(1983)andmore

recentlybyMoranandcolleagues(2007)contradictsthesefindings,astherateofincreaseinTre

andHRduringthefirst20-30minwasconsiderablydifferentbetweenthoseindividualsdeemed

heatintolerantandthoseheattolerant.Thisevidencesuggeststhatitmaybeplausibletoassess

anindividual’sabilitytowithstandexerciseheatstressin30-min.AshorterRHTTrequiringnoprior

testingwouldprovideamoretimeefficientscreeningprocedureforrunners.

To assess and monitor changes in heat tolerance a protocol needs to be reliable to minimise

measurementerror,duetobiologicalvariationandequipmentnoise(AtkinsonandNevill,1998).

Typically,theassessmentofrepeatabilityusesperformancemarkersmoreoftenthanphysiological

markers,andespeciallythermoregulatorymarkers.Whentherepeatabilityofphysiologicalmarkers

has been assessed, it has often been between two pieces of equipment measuring the same

4 Compensableheat stress refers to a steady state Tc,whereby theheat lossmatches theheatproduction. Uncompensable heat stress refers to an imbalance between the evaporativerequirementandtheevaporativecapacityofanindividual.

98

physiological variable. Consequently, there is limited evidence comparing physiologicalmarkers

during repeated trials using a set intensity exercise protocol. The repeatability of mean aural

temperatureandmeanHRduringafixedintensitycyclingheatstresstest,whichinvolvedthree20-

mincycleboutsseparatedby8-minrestwasassessedinadolescents(Brokenshireetal.,2009).ICC

of0.58and0.95formeanauraltemperatureandmeanHR,respectivelyandaTE(CV%)of0.1%

and3%formeanauraltemperatureandmeanHR,respectivelywerereportedandareindicativeof

strongmeasurementrepeatability.Determiningtherepeatabilityofphysiologicalmeasuresduring

aHTTwouldprovideconfidenceintervalsandallowthetesttobeusedtodetermineachangein

heattolerancefollowingacuteorchronicheat-alleviatinginterventions.Thus,itmayenablespecific

guidanceonpreparationrequiredpriortotrainingorcompetinginhighambientconditions.

WhileacknowledgingthelimitationsoftheIDFHTT,pilotdata(3.16.)establishedsimilarpatterns

ofheattoleranceandheatintoleranceprovidingsupporttoexaminetherepeatabilityoftheRHTT.

Thisstudyaimedtoestablishtherepeatabilityofapracticalrunningtesttoevaluateindividuals’

heattolerance.TheprimarilyhypothesiswasthatphysiologicalresponsesduringtheRHTTwillhave

astrongpositivecorrelationonrepeatedtrials.Thesecondaryhypothesiswasthattherewillbeno

differences inphysiological responsesduring the runningheat tolerance testbetween repeated

trials.

4.3. Materialsandmethods

Participants

Sixteen(8males;8females)athletes(3.2.)volunteeredandprovidedwritteninformedconsentto

participateinthestudy(mean±SD;age,23±5yrs.;BM,67.07±10.96kg;stature,1.76±0.10m;

BSA,1.82±0.19m2;sumoffourskinfolds,43±15mm;speedatlactatethreshold,11.7±1.8km.hr-

1;andrunning,VgO2peak49±7mL.kg-1.min-1).ThestudywasapprovedbytheUniversityofBrighton

Research Ethics & Governance Committee and conducted in line with the health and safety

guidelines(3.1.)andinaccordancewiththeguidelinesoftherevisedDeclarationofHelsinki,2013.

Preliminarytesting

During the first visit to the laboratory standardised anthropometric assessmentwas conducted

(3.5.)followedbyagradedexercisetesttodetermineparticipants’lactatethresholdandVgO2peak

(3.15.).Breath-by-breathexpiredairwasmeasuredusingonlinegasanalysisthroughouttheRHTT

(3.9.).Acapillary lactatesample(3.11.)wastakeoncompletionofeachstageduringthelactate

thresholdtestandoncompletionoftheVgO2peaktest.

99


All trials were conducted with pre-trial diet and exercise standardisation (3.4.). Participants

providedaurinesampleonarrivaltoensureadequatehydration.Thetimingoftestingforfemale

participants was conducted in accordance with the menstrual cycle standardisation guidelines

(3.6.). Fiveparticipantswereusingoral contraceptive. twousedYasmin (30µgethinylestradiol/

3mgdrospirenone)andthreeusedMicrogynon(30µgethinylestradiol/150µglevonorgestrel).All

testingwasperformedonamotorisedtreadmill(3.15.).

TheRHTT (3.16.)wasperformedon twooccasions. Following a 20-min stabilisationperiod in a

temperateenvironment(3.7.),measuresofTre(3.8.),HR(3.9.),Tsk(3.8.),RPE,andTS(3.13.)were

recorded,andparticipantsenteredtheenvironmentalchamber(3.7.).ToweldriednudeBMwas

alsorecordedtocalculateSWR(3.12.).ThroughouttheRHTTTre,HR,andTskwererecordedat5-

minintervals.RPEandTSwererecordedat10-minintervals.Exercisewasterminatedinlinewith

universityguidelines(3.1.).Participantsrepeatedtheprocessbetween5-7dlatertoassessthe

repeatabilityoftheRHTT.MeanTsk(Equation2.4)andPSIweresubsequentlycalculated(Equation

3.2).

Statisticalanalyses

AlldatawerefirstcheckedfornormalityusingtheShapiro-Wilkmethod.Physiologicalmeasures

duringRHTT1andRHTT2wereexaminedusingabatteryofrepeatabilitystatistics (3.17.3).Asa

measureofretestcorrelation,ICCwith95%CIwascalculatedforeachvariable.TEMwascalculated

andexpressedasapercentageofitsrespectivemeantoformtheTE(CV%).Bland-Altmanlimitsof

agreement(LOA)plotsshowingthemeanbiasand95%CIwereproduced.Theindividualparticipant

differences,betweenthetwotrialsforeachvariablewereplottedagainsttherespectiveindividual

means.Pairedsampledttestswerecalculatedtoidentifyanydifferencesinphysiologicalmeasures

between RHTT1 and RHTT2 (3.17.4). Effect sizes (Cohens’ d) were calculated to analyse the

magnitude of the interaction (Lakens, 2013) (3.17.5). All data were analysed using a standard

statistical package (SPSS version 20.0), and reported asmean ± SD. Statistical significancewas

acceptedatthelevelofp≤0.05.

4.4. Results

MeasuresofICC,TEM,theTE(CV%)andmeanbiaswithLOAforkeymarkersofheattoleranceare

presentedintable4.1.StrongcorrelationspresentedasICCwereobservedinpeakTre(r=0.93),

peakTsk(r=0.95),peakHR(r=0.99),peakPSI(r=0.98),andSWR(r=0.95)betweenRHTT1and

100

RHTT2(figure4.1).TheseobservedsimilaritiesarefurthersupportedbyalowTEMandTE(CV%)

forthekeymarkersofheattolerancebetweenRHTT1andRHTT2.

Figure 4.2 demonstrates small mean bias and LOA between trials for physiological measures

betweenthetwotrials.Inaddition,therewerenodifferencesobservedinpeakTre(t(15)=-0.785,p

=0.445,d=0.20),peakTsk(t(15)=0.223,p=0.827,d=0.06),HRpeak(t(15)=-1.005,p=0.331,d=

0.25),peakPSI (t (15)= -0.969,p=0.348,d=0.24),andSWR(t (15)=0.087,p=0.931,d=0.02)

betweenRHTT1andRHTT2.

StrongcorrelationspresentedasICC(95%CI)wereobservedinTrerest(r=0.88(0.38,0.90),p≤

0.001),Tremean(r=0.92(0.77,0.97),p≤0.001),HRrest(r=0.91(0.76,0.97),p≤0.001),andHR

mean(r=0.97(0.98,0.99),p≤0.001)betweenRHTT1andRHTT2.Theseobservedsimilaritiesare

furthersupportedbyalowTEMandTE(CV%)forTrerest(0.17°C,0.45%),Tremean(0.15°C,0.39%),

HRrest(3beats.min-1,5%),andHRmean(3beats.min-1,2%).ThereweresmallmeanbiasandLOA

forTrerest[-0.04,(-0.50,0.42)],Tremean[-0.04,(-0.45,0.37)],HRrest[0,(-8,8)],andHRmean[1,

(-8,10)].Furthermore,therewerenodifferencesinTrerest,(t(15)=-0.722,p=0.481,d=0.18)Tre

mean,(t(15)=-0.770,p=0.453,d=0.19)HRrest(t(15)=-0.301,p=0.768,d=0.08),andHRmean

(t(15)=-1.003,p=0.332,d=0.25)betweenRHTT1andRHHT2.

Strongcorrelations(ICC)wereobservedforpeakRPE(r=0.96,(0.88,0.99),p≤0.001)andpeakTS

(r=0.92,(0.76,0.97),p≤0.001)betweenRHTT1andRHTT2.Theseobservedsimilaritiesarefurther

supportedbyalowTEMandTE(CV%)forpeakRPE(0.6,4%),andpeakTS(0.2,3.2%).Therewere

smallmeanbiasandLOAforpeakRPE[-0.1,(-1.7,1.6)],peakTS[0.1,(-0.6,0.7)]betweenRHTT1

andRHTT2.Furthermore,therewerenodifferencesinpeakRPE(t(15)=-0.293,p=0.774,d=0.08)

andpeakTS(t(15)=0.145,p=0.0.27,d=0.28)betweenRHTT1andRHHT2.

101

Table4.1RepeatabilitystatisticsforphysiologicalvariablesduringrepeatedtrialsoftheRHTT.Mean±SD.

Trepeak(°C) Tskpeak(°C) HRpeak(beats.min-1) PSIpeak SWR(g.hr-1)

RHTT1 38.82±0.47 38.12±0.45 182±15 8.7±1.5 1,721±675

RHTT2 38.86±0.49 38.11±0.45 183±15 8.8±1.5 1,716±745

ICC(95%CI) 0.93(0.89,0.99);p≤0.001 0.95(0.85,0.98);p≤0.001 0.99(0.96,0.99);p≤0.001 0.98(0.94,0.99);p≤0.001 0.95(0.86,0.98);p≤0.001

TEM 0.13 0.14 2 0.3 162

TE(CV%) 0.34 0.37 1 3.0 9

Meanbias(LOA) -0.04(-0.41,0.33) 0.01(-0.38,0.40) -1(-8,6) -0.1(-0.9,0.7) -133(-492,227)

Notes:HR,heartrate;ICC(95%CI),intraclasscorrelationcoefficientwith95%confidenceintervals;LOA,limitsofagreement;PSI,physiologicalstrainindex;RHTT,runningheattolerance

test;TEM,typicalerrorofmeasure;TE(CV%),typicalerrorasacoefficientofvariation;Tre,rectaltemperature;Tsk,skintemperature;SWR,sweatrate

102

Figure4.1 Peak rectal temperature (Tre),peakheart rate (HR), sweat rate (SWR),andpeak skin

temperature(Tsk)duringRHTT1(x-axis)andRHTT2(y-axis),formales(closedmarkers)andfemales

(openmarkers).Dottedlinerepresentslineofequality.N=16(8M,8F).

38.0

38.5

39.0

39.5

40.0

38.0 38.5 39.0 39.5 40.0

PeakT

re(°C)RHT

T2

PeakTre (°C)RHTT1

140

150

160

170

180

190

200

210

140 150 160 170 180 190 200 210

PeakHR(beats.m

in¯¹)R

HTT2

PeakHR(beat.min¯¹)RHTT1

500

1000

1500

2000

2500

3000

3500

4000

500 1000 1500 2000 2500 3000 3500 4000

SWR(g.hr¯¹)RH

TT2

SWR(g.hr¯¹)RHTT1

37.0

37.5

38.0

38.5

39.0

37.0 37.5 38.0 38.5 39.0

PeakT

sk(°C)RHT

T2

PeakTsk (°C)RHTT1

Males

Females

103

Figure4.2Bland-Altmanplotswithmeanbias(solidline)and95%limitsofagreement(dottedline)

for peak rectal temperature (Tre), peak heart rate (HR), sweat rate (SWR), and peak skin

temperature(Tsk)formales(closedmarkers)andfemales(openmarkers).N=16(8M,8F).

4.5. Discussion

TheprincipleaimofthecurrentstudywastoexaminetherepeatabilityofaRHTTfromtwotrials

separatedby5-7d.ClassicmarkersofheattoleranceandHA,namelypeakTre,peakTsk,peakHR,

andSWRwereusedforanalysis(Moranetal.,2007;Sawkaetal.,2011a).Themainfindingfrom

-0.7

-0.5

-0.3

-0.1

0.1

0.3

0.5

0.7

38.0 38.5 39.0 39.5 40.0

Diffe

renceinpeakT r

ebe

tweentrials(°C)

MeanpeakTre betweentrials(°C)

-11

-9

-7

-5

-3

-1

1

3

5

7

9

11

150 170 190 210

Diffe

renceinpeakHR

betweentrials

(beats.m

in¯¹)

MeanpeakHRbetweentrials(beats.min¯¹)

-600

-400

-200

0

200

400

600

0 1000 2000 3000 4000

Diffe

renceinSWRbe

tweentrials(g.hr¯¹)

MeanSWRbetweentrials(g.hr¯¹)

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

37.0 37.5 38.0 38.5 39.0

Diffe

renceinpeakT s

kbe

tweentrials(°C)

MeanpeakTsk betweentrials(°C)

Males

Females

104

thecurrentstudyisthattheRHTThadgoodagreement,strongcorrelationsandsmalldifferences

between repeated trials and the TEM values for these classic markers suggested low within-

participantvariability.Thesefindingsareinsupportoftheaimofthestudy.Specifically,theprimary

andsecondaryhypotheseswerebothsupported.ValuesofpeakTre,peakTsk,peakHR,andSWR

spreadalongacontinuum,withawiderangeof responses,challengingthepreviousperception

thatindividualsareeitherheatheattolerantorheatintolerant(Kresfelderetal.,2006;Moranet

al., 2007). The data presented in the current study, demonstrates that the RHTT has strong

repeatabilityandisabletodifferentiatebetweenindividualresponsestotheRHTT.Thesefindings

supporttheuseoftheRHTTinfutureinvestigationstogaugeindividual’srunningheattolerance,

andtomonitortheextentofacuteandchronicheat-alleviatingprotocols.

To the author’s knowledge, there are no studies assessing the repeatability of physiological

measures during a fixed intensity running protocol in the heat, making acceptable levels of

repeatability a priori difficult to determine. Furthermore, there is no literature assessing the

repeatabilityoftheclassicalIDFHTTwhichwouldoffercomparison.Asageneralrule,acorrelation

coefficientover0.90isconsideredtobehigh,between0.70–0.80moderate,andbelow0.70tobe

lowforphysiologicaltests(Vincent,1995). Inthecurrentstudy,ICCvaluesforpeakTre,peakTsk,

peakHR,peakPSI,andSWRwereallequalorgreaterthan0.93.Basedonthesepredetermined

criteria, it is reasonable to suggest that the physiological measures during the RHTT have an

acceptablelevelofrepeatabilitybetweenrepeatedtrialsbasedonICC.

Thedata inthecurrentstudydemonstrates lessvariabilitythanavailabledataonfixedintensity

cyclingprotocolsintheheatusingsimilarphysiologicalvariables.TheTE(CV%)of0.3%and1%for

peakTreandpeakHRrespectively,comparefavourablytotheTE(CV%)of0.3%and3.9%formean

TreandmeanHRreportedduringa60-min,fixedintensitycyclingprotocolinhothumidconditions

(Haydenetal.,2004).Furthermore,Brokenshireandcollegues(2009)investigatedtherepeatability

ofHR during a fixed intensity cycling heat stress test,which involved three 20-min cycle bouts

separatedby8-min rest. The ICCof0.95andTE (CV%)of3% formeanHRwere reportedand

assumed indicative of strongmeasurement repeatability. The ICC of 0.99 and TE (CV%) of 1%

reportedinthecurrentstudy,againcomparefavourablytothesefindingshighlightingthestrength

oftheRHTTintermsoflowmeasurementerror.

To aid interpretation and presentation of results when assessing individuals’ heat tolerance,

researchers havedichotomized the population and categorized individuals as either tolerant or

intolerant(Kresfelderetal.,2006;Moranetal.,2007).AltmanandRoyston(2006)reportedseveral

problemswithdichotomizingdata.Statisticalpowertodetectarelationshipmaybereduced,there

105

isanincreasedriskofreportingafalsepositive,apossibilityofunderestimatingtheextentofthe

variationintheoutcomebetweengroups,andusingtwogroupsconcealsanynon-linearityinthe

relationship.AltmanandRoyston(2006)stateusingmultiplecategories isgenerally favouredto

dichotomizingdata;withfourorfivegroupsthelossofinformationcanbequitesmallbutthere

arecomplexitiestoanalysis.Indeed,Moranandcolleagues(2007)acknowledgethatthelargerthe

deviationsfromnormalvaluesthemorepronouncedthestateofheatintolerance,thusimplyinga

continuum of heat tolerance. Similarly work from, Taylor and Cotter (2006) propose that heat

adaptation isa continuum,with thepositionofan individualalong thecontinuumrepresenting

progressiveincreasesinheattolerance.Thefindingsinthepresentstudy,demonstrateclearlythat

anindividual’sheattolerance,representedbythepeakTre,peakTsk,peakHR,peakPSI,andSWRis

acontinuousvariable;demonstratingthatheattolerancemaybemoreaccuratelycategorisedon

a continuum. Consequently, these findings support the use of the RHTT to track changes in

individuals’ ability to withstand exercise heat stress from acute and chronic heat-alleviating

interventions.

Casesofexertionalheat illness canoccuramongenduranceathletes, inextremecircumstances

leading to death; however, the epidemiology is not well documented in the literature.Martin

(1997)reportedthehighestincidenceofexertionalheatillnessoccurredinlongdistancerunners

duringthe1992NewOrleansU.S.OlympicTrialsandthe1996AtlantaOlympics;withlongdistance

runners accounting for 31% and 53% respectively, of the total cases of exertional heat illness.

Furthermore,Nielsen(1996)providesdatatosuggesttheincidenceofheatillnessisunavoidable

forendurance runnerswhencompeting inahighambient temperaturecombinedwithhighRH

withoutasevere reduction inenduranceperformance;highlighting the importanceof thorough

preparationtopreventtheincidenceofaheatillness.Thefindingsinthecurrentstudycouldbe

applied in a manner that would serve to minimise the number of athletes that suffer from

hyperthermia, by supporting amore complete evaluation and subsequent preparation prior to

trainingandcompetingintheheat(Johnsonetal.,2013).

TheTEMexpressedas a coefficientof variation canbeused toestimate sample size for future

studiesusingtheRHTTwhenasmallestworthwhilechangeisknown;usingtheformulaN=8s2/d

2

(where‘s’=typicalerrorexpressedasacoefficientofvariationand‘d’=thesmallestworthwhile

change (Hopkins, 2000b). A change of 0.4°C typically represents a significant reduction in Tre

followingacuteandchronicheat-alleviatingprocedures(Buonoetal.1998;Pattersonetal.2004;

Castleetal.2006;Garrettetal.2009).Accordingly,inthecurrentstudytodetecta0.4°Cchange,a

sample sizeof sixparticipantswouldbesufficientwhenpeakTre is thekeyvariableof concern,

assumingsimilarvariabilityamongtheparticipantsrecruited.

106

4.6. Limitations

The RHTT adopts a fixed absoluteworkload,which is an appropriate procedure to assess heat

tolerance when the experimental design is a repeated measure. However, the fixed absolute

workloadmaylimitapplicabilityofthetestwhencomparingbetweengroups,especiallywhenthey

are not matched. The recent work by Cramer & Jay (2014) reports when comparing between

individuals or groups unmatched from a biophysical perspective, exercise intensity should be

administeredusingfixedheatproductionrelativetoBMtopreventtheintroductionofsystematic

bias. Future research is warranted to quantify the potential differences in metabolic heat

production between participants of differing BM, BSA and fitness during the RHTT to ensure

unbiasedcomparisonofthermoregulatoryresponsesbetweenindividuals(CramerandJay,2014).

Furthermore,theroleoftheRHTTinidentifyingindividuals’susceptibilitytoexertionalheatillness

hasnotbeen identifiedwithin the current study.CollectingRHTTdataon individualswhohave

previously incurred exertional heat illness would test the value of the RHTT in identifying an

individual’ssusceptibilitytoexertionalheatillness.

4.7. Conclusion

ThemainfindingfromthecurrentstudywasthattheRHTTdemonstratedgoodagreement,strong

correlationsandsmalldifferencesbetweenrepeatedtrials.TheTEMvaluesshowed lowwithin-

participantvariability. Inaddition,data ispresentedtodemonstratethattheRHTT iscapableof

differentiating between individuals’ responses to the RHTT when adopting a running mode of

exercise;providingevidencethata30-minHTTprotocolissufficientinduration.Furthermore,the

findingsfromthepresentstudychallengethepreviousreportsthatheattoleranceisdichotomous,

sinceindividualresponseswerelinearlyspreadoveracontinuum.Thesefindings,couldbeusedto

observeindividuals’thermoregulatoryresponsesovertime,providinginformationtophysiologist,

coaches,andmedicalstafftoassistdecisionsaboutathletes’safetypriortotrainingandcompeting

intheheat.Inaddition,theRHTTcouldbeusedasatoolforinvestigatingtheeffectofacuteand

chronicheat-alleviatingprocedures.

ThischapterexploredtherepeatabilityoftheRHTT.Thisestablishedituseasameasurementtool.

Chapter5(Study2)willassessthesensitivityoftheRHTTtochangesinheattolerance.

107

5. Sensitivityofarunningheattolerancetest

________________________________________________________________________________

Study1(Chapter4)ofthisthesishasestablishedtheRHTTasreliable,modespecifictest.Toassess

thesensitivityoftheRHTTtochangesinheattolerance,datawaspooledfromtwofurtherstudies

ofwhicharenotpresentedelsewhereinthethesis.

5.1. Abstract

EstablishingwhethertheRHTTissensitivetochangesinheattoleranceisessentialtosupportfuture

use of the RHTT in providing a more complete evaluation of athletes’ heat tolerance, and to

quantifying the effectiveness of heat-alleviating procedures. This study aimed to identify the

sensitivity of the RHTT to changes in heat tolerance following STHA. Twenty-five (12M, 13F)

participantsperformedtwoRHTT,24-hrprecedingHA(RHTT1)and24hrsfollowing5-dcontrolled

hyperthermia(Tre=38.5°C)HA(RHTT2).TheRHTTinvolved30-minrunning(9km.hr-1,2%gradient)

in40°C,40%RH(WGBT=33°C).FollowingHA,valuesofpeakTre(RHTT1:38.94±0.48°C,RHTT2:

38.56±0.55°C,p≤0.001),peakTsk(RHTT1:37.47±0.49°C,RHTT2:37.06±0.70°C,p=0.003),and

peakHR(RHTT1:185±14beats.min-1,RHTT2:175±14beats.min

-1,p≤0.001)reduced,andSWR

increased(RHTT1:1,070±541g.hr-1,RHTT2:1,481±457g.hr

-1,p=0.003).Therangeofreductions

inmeasuresofpeakTre(-0.48°C,-0.03°C),peakTsk(-1.74°C,0.13°C),peakHR,(-24beats.min-1,-2

beats.min-1)andSWR(-460g.hr

-1,1640g.hr

-1)demonstratemarkedindividualdifferences.Thedata

suggeststheRHTTissensitivetochangesinheattoleranceandthatchangesinheattoleranceare

highlyindividual.

5.2. Introduction

Duringprolongedsubmaximalexerciseinahotenvironmentthereisariseinbodyheatstorage,

which in turn reflects thebalancebetweenmetabolicheatproduction,heatabsorbed fromthe

environmentandtotalbodyheatloss(JayandKenny,2007).Thereisawidevariationinindividual

abilitytowithstandexerciseheatstress(Epstein,1990).Underextremeconditionsofexertionin

theheatevenhealthy,heatacclimated,andendurancetrainedindividualswillincreasetheirbody

heat content. Individualswhoexperienceagreater storageofmetabolicheatdue toa reduced

sweatingsensitivityanda lowadaptivecapacitytoexercise intheheat,havebeendescribedas

heatintolerant(Epsteinetal.,1983;Moranetal.,2004).Theabilitytoquantifyanindividual’slevel

108

ofheattoleranceoffersvaluableinformationtosupportthepreventionoffutureoccurrenceofa

heat-relatedillness.

A RHTT was developed in Study 1 (Chapter 4). The protocol involves 30-min of running on a

treadmill(9km.hr-1anda2%gradient)inambientconditionsof40°Cand40%RH(WBGT=33°C).

Classicmarkersofheattoleranceandadaptationnamely,Tre,Tsk,HR,andSWRdemonstratedgood

agreement, strong correlations, (ICC>0.93) and small differences (p ≥ 0.05)between repeated

trials. Furthermore, the TEM values for these classicmarkers suggested lowwithin participant

variability.TheRHTTdemonstrateddifferentdegreesofheattolerancebetweenindividualswhich

reinforcesthattheRHTTisanappropriatetooltoevaluateheattolerance.Thereiscurrentlyno

evidence supporting the sensitivity of the RHTT to changes in heat tolerance. Establishing the

sensitivityoftheRHTTtochangesinheattolerancemaysupporttheuseoftheRHTTtomonitor

changesinheattolerancefollowingacuteandchronicheat-alleviatingprocedures.Inaddition,it

maysupporttheuseoftheRHTTtomonitorchangesinheattolerancethatoccurnaturallytohelp

identifyperiodsoftimewhenathletesareexperiencingperiodsoflowheattolerancetosupport

thepreventionofdevelopingaheat-relatedillness.

An individual’sheat tolerancehasbeendefinedas theability towithstandexerciseheat stress,

combinedwiththeirabilitytoadapttoexercisingintheheat(Shapiroetal.,1979).Routinely,HTTs

are used in isolation to assess heat tolerance following a heat illness incident to support the

individual’sreturntotraining.Recently,Johnsonandcolleagues(2013)providedcasestudydatato

supporttheuseofacombinedheattolerancescreeningprocedure,usingbothacuteandchronic

heatexposuretoassessatriathlete’sexerciseheattolerance.Theseexploratoryfindingssupporta

combined method to assess athletes’ heat tolerance to facilitate optimal preparation prior to

trainingandcompetingintheheat.

ThisstudyaimedtoestablishthesensitivityoftheRHTTtochangesinheattolerance.Furthermore,

this studyaimed toexplore the individual changes inheat tolerance followingHA. Theprimary

hypothesiswasthattherewillbedifferencesinphysiologicalresponsesduringtheRHTTfollowing

STHA.ThesecondaryhypothesiswasthattheRHTTwillbesensitivetoindividualdifferencesinthe

magnitudeofadaptation.

109


Participants

Datawas pooled from two data set collected at different time periods that are not presented

elsewhere inthethesis.Twenty-five(12M;13F)athletes(3.2)volunteeredandprovidedwritten

informedconsenttoparticipate(table5.1).ThestudywasapprovedbytheUniversityofBrighton

Research Ethics & Governance Committee and conducted in line with the health and safety

guidelines(3.1.)andinaccordancewiththeguidelinesofthereviseddeclarationofHelsinki,2013.

Preliminarytesting


(3.5.). A graded exercise test was performed to determine participants’ VnO2 peak on a cycle

ergometer(3.14.).Breath-by-breathexpiredairwasmeasuredusingonlinegasanalysis(3.9.)and

acapillarylactatesamplewastakenoncompletionofthetest(3.11.).Aregressionequationwas

computed from the data obtained to calculate the required intensity (65% VnO2 peak) for the

experimentalcontrolledhyperthermiaHAexercisesessions(Appendix4).

Table5.1Participantcharacteristics.Mean±SD(ranges).

BM

(kg)

Stature

(m)

BSA

(m2)

Sumof4skin

folds(mm)

VSO2peak

(mL.kg-1.min-1)

AllData(N=25)65.67±10.93

(41.30-89.42)

1.71±0.01

(1.46–1.89)

1.76±0.19

(1.30-2.07)

38±11

(20–56)

48±8

(33–69)

Males(N=12)74.13±7.28

(64.70-89.42)

1.78±0.06

(1.72–1.89)

1.92±0.10

(1.79-2.07)

32±8

(20–46)

51±8

(38–69)

Females(N=13)57.86±9.21

(41.30-75.00)

1.64±0.09

(1.46-1.78)

1.62±0.17

(1.30-1.90)

43±11

(22–56)

45±7

(33–56)

Notes:BM,bodymass;BSA,bodysurfacearea;VnO2peak,peakoxygenuptake

110

Experimentaldesign

Testing was completed over a 7-d period. The timing of testing for female participants was

conducted in line with the menstrual cycle standardisation guidelines (3.6.). Five female

participantswere using oral contraceptive: three usedMicrogynon, one used Rigevidon (30 µg

ethinylestradiol/ 150 µg levonorgestrel) and one used Yasmin (30 µg ethinylestradiol/ 3mg

drospirenone). All trialswere conducted in linewith pre-trial diet and exercise standardisation

(3.4.). Participants provided a urine sample upon arrival to ensure adequate hydration (3.4.).

VolunteersperformedfivecontrolledhyperthermiaHAsessionsonacycleergometer(3.14.)and

twoRHTTs(3.16.).ThefirstRHTTwasperformed24-hrpriortobeginningHA(RHTT1),thesecond

RHTTwasperformed24-hrfollowing5-dHA(RHTT2).Allexperimentaltrialswereperformedina

hotenvironment(3.7.).Exercisewasterminatedinlinewithuniversityguidelines(3.1.).Therewere

noincidencesofearlyterminationofexerciseduringtheRHTTwithinthisstudy.


TheRHTT(3.16.)wasperformedontwooccasionsonamotorisedtreadmill(3.15.).Followinga20-

minstabilisationperiodinatemperateenvironment(3.7.),measuresofTre,Tsk(3.8.),HR(3.9.),RPE,

andTS(3.13.)wererecorded.Theparticipantsthenenteredtheenvironmentalchamber.Towel

driednudeBMwasalsorecordedtocalculateSWR(3.12.).ThroughoutthetestTre,Tsk,andHRwere

recordedat5-minintervals.RPEandTSwererecordedat10-minintervals.MeanTsk(Equation2.4)

andPSI(Equation3.2)weresubsequentlycalculated.

Statisticalanalyses

DatawereassessedfornormalityusingtheShapiro-Wilkmethod.Apairedsamplettest(3.17.4)

wascalculatedtoidentifyanydifferencesinphysiologicalmeasuresbetweenD1andD5ofHAand

betweenRHTT1andRHTT2.Effectsizes(Cohen’sd)werecalculatedtoanalysethemagnitudeof

theinteraction(Lakens,2013)(3.17.5.).Alldatawereanalysedusingastandardstatisticalpackage

(SPSSversion20.0),andreportedasmean±SD.Statisticalsignificancewasacceptedatthelevelof

p≤0.05.

111

5.4. Results

Heatacclimation

Allparticipantscompletedfiveconsecutivedaily90-minHAsessions.Participantsmaintainedthe

targetTreof38.5°Conaveragefor48±9minduringeachsession.Themeandurationofexercise

was64±12minpersession.Themeansessionintensity(includingrestperiods)was41±4%VnO2

peak;however, themeanexercise intensity (excludingrestperiods)was58±7%VnO2peak.The

meanTreandHRduringeachHAsessionwas38.27±0.13°Cand147±9beats.min-1,respectively.

Furthermore,themeanSWRduringeachHAsessionwas556±153g.hr-1.

TherewasareductioninTrerest(t(24)=2.192,p=0.037,d=0.408)andHRrest(t(24)=3.758,p≤

0.001,d=0.609)fromD1toD5ofHA.TherewasanincreaseinSWRduringtheHAsessionfrom

D1toD5(t(24)=-3.030,p=0.006,d=0.526).Furthermore,therewasanincreaseinSWRBSA(t(24)=

-2.917,p=0.008,d=0.512).


Table5.2presentsthemean±SDdataforTrerest,peakTre,peakTsk,HRrest,peakHR,andSWR

duringtheRHTT.Therewasa0.16±0.29°Cand0.38±0.38°CreductioninTrerest(t(24)=2.517,p=

0.019,d=0.46)andpeakTre(t(24)=4.957,p≤0.001,d=0.71)respectively,fromRHTT1toRHTT2.

Adaptive responsevariedbetweenparticipantswith changes inTre rest ranging from -0.62°C to

0.41°CandpeakTrerangingfrom-1.43°Cto-0.03°C.Therewasa0.41±0.51°CreductioninpeakTsk

fromRHTT1andRHTT2(t(24)=3.348,p=0.003,d=0.56);changesrangedfrom-1.74°Cto0.93°C

(figure5.1).

Therewasa4±9beats.min-1and10±9beats.min

-1reductionsinHRrest(t(24)=2.195,p=0.038,

d=0.41)andpeakHR(t(24)=5.788,p≤0.001,d=0.76)respectively,fromRHTT1toRHTT2.Adaptive

responsesvariedbetweenparticipantswithchangesinHRrestrangingfrom-19beats.min-1to8

beats.min-1andpeakHRrangingfrom-24beats.min

-1to0beats.min

-1.

ThesechangesresultedinanaveragereductioninpeakPSIfromaveryhighstraintoahighstrain

(Moran et al., 1998) (t (24) = 5.268, p < 0.001, d = 0.73). Adaptive responses varied between

participantswithchangesinPSIpeakrangingfrom-0.06to4.30.

112

Table5.2PeakphysiologicalvariablesduringtheRHTT.Mean±SD(ranges).

RHTT1 RHTT2 Difference

Trerest(°C) 37.17±0.32 37.01±0.34* -0.16(-0.65,0.41)

Trepeak(°C) 38.94±0.48 38.56±0.55* -0.38(-0.48,-0.03)

Tskpeak(°C) 37.47±0.49 37.06±0.70* -0.41(-1.74,0.13)

HRrest(beats.min-1) 69±10 65±9* -4(-28,15)

HRpeak(beats.min-1) 185±15 175±14* -10(-24,-2)

SWR(g.hr-1) 1,070±541 1,481±457* +411(-460,1640)

SWRBSA(g.hr-1.m2) 597±272 797±225* +200(-459,793)

Notes:HR,heartrate;RHTT,runningheattolerancetest;SWR,sweatrate;Tre,rectaltemperature;Tskskin

temperature.*denotessignificantdifferencebetweenRHTT1andRHTT2(p≤0.05).

Therewasa411g.hr-1increaseinSWR(t(24)=-3.328,p=0.003,d=0.56)fromRHTT1toRHTT2;

changesrangedfrom-460g.hr-1to1640g.hr

-1.Furthermore,therewasanincreaseinSWRBSA(t(24)

=-3.505,p=0.002,d=0.58)fromRHTT1toRHTT2.

TherewasareductioninpeakRPE(-2;t(24)=4.797,p≤0.001,d=0.700)andpeakTS(-0.5;t(24)=

2.771,p≤0.001,d=0.492)fromRHTT1toRHTT2.

113

Figure5.1Individualdataforvaluesofpeakrectaltemperature(Tre),peakskintemperature(Tsk),

peakheatrate(HR)andsweatrateSWRduringRHTT1(xaxis)andRHTT2(yaxis)formales(closed

markers)andfemales(openmarkers).Dottedlinerepresentslineofequality.N=25(12M,13F).

37.5

38.0

38.5

39.0

39.5

40.0

37.5 38.0 38.5 39.0 39.5 40.0

PeakT

re(°C)RHT

T2

PeakTre (°C)RHTT1

35.0

36.0

37.0

38.0

39.0

35.0 36.0 37.0 38.0 39.0

PeakT

skRH

TT2

PeakTsk RHTT1

Males

Females

0

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

SWR(g.hr¯¹)RH

TT2

SWR(g.hr¯¹)RHTT1

Males

Females130

140

150

160

170

180

190

200

210

130 140 150 160 170 180 190 200 210

PeakHR(beats.m

in¯¹)R

HTT2

PeakHR(beats.min¯¹)RHTT1

114

5.5. Discussion

ThemainfindinginthecurrentstudywasthattheRHTTissensitivetothechangesinheattolerance

thatareassociatedwithSTHA,evidencedbyareductioninTre,Tsk,andHR,andanincreaseinSWR.

Thesefindingssupporttheaimofthestudy.Specifically,theprimaryandsecondaryhypotheses

were both supported. These results support the use of the RHTT to monitor changes in heat

tolerancewhenusingarepeatedmeasuresdesign.Furthermore,thedatademonstratesmarked

differencesbetweenparticipants’changesinheattolerance,suggestingheatadaptationishighly

individual.

Heatadaptationresponses

Therewasa0.38°CmeanreductioninpeakTrefollowingSTHAinthecurrentstudy.Study1(Chapter

4)identifiedtheTEMforpeakTreduringtheRHTTtobe0.13°C,suggestingatruereductionof>

0.25°C. These findings, are comparable to the~0.24°C reduction, previouslyobserved following

STHA using a controlled hyperthermia model (Garrett et al., 2009; Patterson et al., 2004a).

Furthermore,Study1(Chapter4)identifiedaTEMof0.14°CforpeakTskduringtheRHTT,the0.41°C

reductionobservedinthecurrentstudyalsosuggestsatruereductionof>0.27°C.Theobserved

reductions inthermoregulatorystrainarepotentiallyduetoanenhancedevaporativeheat loss,

thus reducing heat storage. These results demonstrate the sensitivity of the RHTT to monitor

changesinthermoregulatorystrainassociatedwithSTHA.

Therewas a 10 beats.min-1mean reduction in peakHRduring the RHTT following STHA in the

currentstudy.Study1(Chapter4)identifiedtheTEMforpeakHRduringtheRHTTtobe2beats.min-

1, suggesting a true reduction of > 8 beats.min

-1. These findings are comparable to the ~12

beats.min-1reductionpreviouslyobservedfollowingSTHAusingacontrolledhyperthermiamodel

(Garrettetal.,2009;Pattersonetal.,2004a).Thereductionincardiovascularstrainrepresentan

improved myocardial efficiency and ventricular compliance (Lorenzo et al., 2010). Due to the

matched exercise intensity of the RHTT, the observed reduction in cardiovascular strain

demonstrates the sensitivity of the RHTT to monitor adaptation to the cardiovascular system

associatedwithSTHA.

Therewasa411g.hr-1meanincreaseobservedinSWRfollowingSTHAinthecurrentstudy.Study

1(Chapter4)identifiedtheTEMforSWRduringtheRHTTtobe162g.hr-1,suggestingatrueincrease

of≥249g.hr-1;thesefindingsarecomparabletothe220g.hr

-1previouslyobservedfollowingSTHA

usingacontrolledhyperthermiamodel(Pattersonetal.,2004a).AnincreaseinSWRcouldbeeither

115

analteredafferentneuralactivityfromtheperipheralorcentralthermo-receptorscausingdifferent

integrationofthermalinformation,analteredefferentneuralactivityforagivenlevelofafferent

inputoranalteredeffectorresponse.Thepotentialmechanismsforthisincludealterationsinthe

Tcthresholdforsweatingonset(Shvartzetal.,1979),anincreasedsweatingsensitivity(Shvartzet

al.,1979)and/oreccrinesweatglandhypertrophy(Satoetal.,1990).Duetothematchedexercise

intensityduringtheRHTT,therepeatedtrialswereassumedtobematchedfortherequirementfor

evaporative heat loss thus, these results demonstrate the sensitivity of the RHTT to monitor

changesinsudomotorfunctionfollowingHA.

Individualresponses

Observing individual heat tolerance using both an acute and chronic heat exposure has been

suggestedtoofferamorecomprehensiveassessmentofindividualheattolerance(Johnsonetal.,

2013).However,themajorityofHAliteraturereportsaverageresponsesandfailstorecognisethe

differences inadaptiveresponses.Thedatademonstrated largedifferences inthemagnitudeof

adaptationbetweenindividuals.Forexample,oneparticipantinthecurrentstudydemonstrateda

largeadaptiveresponsefollowingSTHAwithpeakPSIreducingfromhigh(8.65)tomoderate(6.98)

strain, combined with a 150% increase in SWR. However, another participant experienced no

alterations in peak PSI and had only a 23% increase in SWR, demonstrating a small adaptive

response.

There are several factorswhich havebeen suggested to alter themagnitudeof the adaptation

response which may explain the variance observed in the current study. Firstly, the closer an

individualistotheirgeneticallydeterminedmaxima,thegreatertheadaptationresistanceasthe

adaptionreservenarrowswithprogressivelysmallgainsoccurringovertime(Prud’Hommeetal.,

1984).Furthermore,withinanygivenpopulationtherearehighrespondersandlowrespondersto

thermal stimulation (Taylor, 2014). The majority of young sedentary individuals who live in a

predominantlytemperateclimatewillbehighthermalresponders.Whereas,individualswholive,

workandtraininhighambientconditionswillshowadaptationresistance(Taylor,2014).

TheexerciseelicitedduringtheHAsessionswasperformedat65%ofVnO2peak.Thereareseveral

limitationsobservedwithsettingexercisepercentagebasedonVnO2peakwhichmayexplainthe

varianceobservedinthemagnitudeofadaptation incurrentstudy.TheVnO2peakvaluesforthe

participants’ in the current study ranged from33mL.kg-1.min

-1 to 69mL.kg

-1.min

-1. Thus, those

individualswithalowerVnO2peakworkedatalowermetabolicheatproductionprovidingalower

stimulusforsweatproduction(CramerandJay,2014;Gagnonetal.,2013,2008)andpotentially

116

constrainingadaptation.TheBMoftheparticipants’inthecurrentstudyrangedfrom41.3kgto

89.4kg.AlowerBMentailslessheatstorageandtherefore,alowerexerciseintensityisrequired

to increaseTc to38.5°C (Gagnonet al., 2009). Consequently, the stress imposedduring theHA

sessionswaslikelylowerinparticipantswithalowerBMcomparedwiththosewithahigherBM,

potentially constraining adaptation and influencing the time course of adaptation due to

inadequate endogenous heat strain. Furthermore, changes in Tc are not only a function of the

changeinbodyheatcontent,butalsoofitsmassandtissuecompositionasdefinedbythespecific

heat of the tissue of the human body. The participants’ sum of skin fold and thus, body fat

percentagerangedfrom20mmto56mm.Anindividualwithalowerbodyfatpercentagewillhave

ahigherspecificheat.Thus,atagivenchangeinbodyheatcontentparticipantswithalowbodyfat

percentage will have a lower change in Tc (Jay and Kenny, 2007), potentially constraining

adaptation.

Johnsonetal.,(2013)observedanimprovedheattolerancefollowing9-dHAinatriathletewho

hadpreviousincidencesofheatillness.Followingthisassessment,theathletewasdeemedsafeto

race,duetoasubstantialimprovementinheattolerance,andwentontocompletetheracewith

nosignsofexertionalheatillness.Thecurrentstudysupportsthismodeltoevaluateindividuals’

heat tolerance since therewere a variety of adaptive responses between individuals. Although

thresholds have not been established, those individuals who demonstrate small adaptive

responses,especiallywhencombinedwithahighphysiologicalstrainduringtheRHTT,will likely

experiencesubstantial strainduringcompetitionandpotentiallyatahigher riskofexperiencing

exertionalheatillness.Thus,whenassessingheattolerance,practitionersshouldconsiderusingthe

RHTTpreandpostaheat-alleviatingproceduresuchasHA.Thismethodisconsideredtoprovidea

more in-depth assessment of heat tolerance with information being provided on the initial

exposure and the adaptive capacity of an individual; to support the modification of future

preparationstrategiespriortotrainingandcompetingintheheat.

5.6. Limitations

TheRHTTmaybe limitedby its applicabilitywhen comparingbetweengroups, especiallywhen

participantsarenotmatchedforbiophysicalcharacteristics.Administeringafixedintensityprotocol

whencomparingbetweengroupswillelicitexerciseatavarietyofrequirementsforevaporative

heatloss,resultinginaninaccurateassessmentofthermoregulatorystrainbetweenparticipants

(CramerandJay,2014;Gagnonetal.,2013,2008).However,theRHTTisanappropriateprocedure

toassessheattolerancewhentheexperimentaldesignisarepeatedmeasure,ortotrackanathlete

overtimepriortotrainingandcompetinginhighambientconditions.

117

Furthermore, the roleof theRHTT toaid inclinicaldecisions followingexertionalheat illness in

athletes,andpredictingindividuals’susceptibilitytoexertionalheatillnessisnotknown.Collecting

dataonindividualswhohavepreviouslyincurredexertionalheatillnesswouldreinforcethevalue

oftheRHTT.Providingdiagnosticandprognosticinformationwouldhelpidentifyindividualswho

are at a greater risk of developing exertional heat illness. Future investigations are required to

gatherthisinformationtoimprovethepreparationofindividualspriortotrainingorcompetingin

theheat;ensuringinterventionsareimplementedtobestpreparetheindividual.

5.7. Conclusion

Inconclusion, this studydemonstrates that theRHTT is sensitive tomonitoradjustments in the

classicmarkersofheattoleranceandadaptationwithreductionsinmeasuresofpeakTre,peakTsk,

peakHR,andSWR.ThesefindingssupporttheuseoftheRHTTinfutureinvestigationstoassess

chronic heat-alleviating interventions. Furthermore, these findings could be used to support

optimalpreparation,withindividualisedstrategiesdevelopedbaseduponanindividual’sinitialheat

toleranceandtheiradaptivecapacity,priortotrainingandcompetingintheheat.TheRHTThas

the potential to be used to track changes in heat tolerance assisting decisionmaking about an

athlete’sabilitytotrainandcompeteinhighambientconditions,whilstadoptingamorefavourable

protocolforendurancerunners,duetotherunningmodeofexerciseadministered.

118

6. Acomparisonofmaleandfemaletemporalpatterningtoshort-andlong-termheatacclimation

________________________________________________________________________________

Study1(Chapter4)andStudy2(Chapter5)haveestablishedtheRHTTasareliable,modespecific

(Study1)andsensitivetest(Study2).Thereisapaucityofdataonfemales’responsestoHA.This

chapterwill provide original data onmales and females’ responses to HA using the RHTT as a

methodtoevaluatechangesinheattolerance.

6.1. Abstract

Thecurrentstudyassessedthesexdifferencesinthermoregulatoryandphysiologicaladaptationto

STHA (5d)andLTHA (10d).Sixteen (8M;8F)participantsperformedthreeRHTT,precedingHA

(RHTT1),followingSTHA(RHTT2)andLTHA(RHTT3).TheRHTTinvolved30-minrunning(9km.hr-1,

2%gradient)in40°C,40%RH(WBGT=33°C).FollowingSTHA,Trerest(Males:-0.24±0.16°C,p≤

0.001;Females:-0.02±0.08°C,p=0.597),peakTre(Males:-0.39±0.36°C,p≤0.001;Females-0.07

±0.18°C,p=0.504),andpeakHR(Males:-14±12beats.min-1,p≤0.001;Females:-5±3beats.min

-

1,p=0.164)reducedinmales,butnotfemales.FollowingSTHA,SWRBSAincreased(428±269g.hr

-

1.m

2,p=0.029)infemales,butnotmales(-11±286g.hr

-1.m

2,p=0.029).FollowingLTHA,Trerest

(Males:-0.04±0.15°C,p=0.459;Females:-0.22±0.12°C,p≤0.01)andpeakTre(Males:-0.05±

0.26°C,p=0.590;Females:-0.41±0.24°C,p≤0.01)reducedinfemales,butnotmales.Following

LTHA,SWRBSAincreasedinmales(308±346g.hr-1.m

2,p=0.029),butnotfemales(44±373g.hr

-

1.m

2, p = 0.733). Males and females responded to STHA; however, females required LTHA to

establishreductionsinthermoregulatoryandcardiovascularstrain.ThesefindingssuggestthatHA

protocolsshouldbedesignedtotargetsexdifferencesinthermoregulationforoptimaladaptation.

6.2. Introduction

Increasingambienttemperatureisknowntohaveadetrimentaleffectonenduranceperformance

(GallowayandMaughan,1997).Duringprolongedsubmaximalexerciseinhighambientconditions,

thereisagreaterrequirementforheatlossduetoeitherarateofheatgainfromtheenvironment,

oralowergradientfordryheatloss,typicallyresultinginagreaterchangeinbodyheatcontent

comparedtotemperateconditions.Manyathletes,soldiers,andmanualoperativesexposedtohigh

ambient conditionsare susceptible toheat illnesses; includingheat cramps,heat syncope,heat

exhaustion, and heat stroke. Prior to a heat illness, individuals vary in their ability to tolerate

119

exerciseheatstress,somedemonstratingadecreasedcapabilitytodissipateheatunderthesame

exerciseheatstress(Epstein,1990).Theseindividualsarecharacterizedbyanearlierandgreater

rise in Tc, greater storageofmetabolicheat, greaterphysiological strain, and reduced sweating

sensitivitywhenexercisingintheheat(Epsteinetal.,1983;Moranetal.,2004).

Malesandfemalesdifferintheirthermoregulatoryresponsestoexercise-heatstress,largelydue

to females having a reduced sudomotor function (Gagnon and Kenny, 2011) thus decreasing

evaporative heat loss capacity,with the resultant increase in physiological strain (Moran et al.,

1999). Ithasbeenshownthatmalesandfemalesdisplaysimilarratesofheatdissipationat low

requirements for heat loss. However, sex differences in sudomotor function have been

demonstratedbeyondacertainrequirementforheatloss(GagnonandKenny,2012).Incontrast,

whenmalesandfemalesdisplaysimilarheatlossforagivenheatproduction,femalesmaydisplay

ahigherchangeinTcduetophysicalcharacteristics(Gagnonetal.,2009;Havenith,2001).These

resultssuggestthatfemalesmayreachhyperthermiclevels inashortertimeperiodthanmales,

consequently females have beenmore frequently diagnosed as heat intolerant comparedwith

males (Druyanet al., 2012), potentiallyputting themat greater riskofobtainingaheat-related

illness.Theobservedsexdifferencesinthermoregulationarenotalwaysevident,butthedifference

maybecomemoreevidentastheheatstressincreases(GagnonandKenny,2012).Furthermore,

hormonal fluctuations associated with the menstrual cycle are suggested to modify central

regulatory mechanisms for thermoregulation (Inoue et al., 2005). Elevated progesterone

concentrations during the luteal phase of themenstrual cycle, have been reported to increase

resting Tc by ~0.34°C, the Tc onset threshold for sweating by 0.29°C, and the Tc threshold for

cutaneousvasodilationby0.23-0.30°C(Inoueetal.,2005).

Heatacclimation improvesheat transfer from thebody’s core to the skinandultimately to the

external environment, serving to attenuate physiological strain and improve exercise capacity

(Lorenzoetal.,2010;Sunderlandetal.,2008). HAreducesheatstorage,partiallyasa resultof

adaptationstothesudomotorfunctioncausingan increase inwholebodyevaporativeheat loss

(Poirier et al., 2015). Additionally, HA increases blood volume preserving stroke volume and

reducingHRatagivenworkload(Franketal.,2001;LorenzoandMinson,2010).

There is a dearth of literature assessing female responses to HA. Previously, the physiological

responses ofmales and females to 10-d fixed intensityHAwere assessedwith females initially

exhibiting lower Tre and HR, despite a lower SWR compared withmales (Avellini et al., 1980).

Following HA, the physiological strainwas similar betweenmales and females, althoughmales

maintained a greater SWR. This study adopted a traditional HA protocol which resulted in a

120

progressivedeclineintheadaptationstimulusoverthedurationofHA.Controlledhyperthermia

ensures consistent potentiating stimuli for adaptation throughout the HA period, eliciting

reductionsinthermalstrainandincreasesinworkcapacityduringbothSTHA(Garrettetal.,2012,

2009)andLTHA(Pattersonetal.,2004a),potentiallypromotingmorecompleteadaptation(Taylor

and Cotter, 2006). It remains unknown the extent to which females adapt to the controlled

hyperthermiamodelofHAandwhethertheirresponsesdiffertothatofmales.

HeatacclimationisoftenseparatedintoSTHA(≤8d)andLTHA(≥10d)(Garrettetal.,2011).STHA

is a preferred regime, as it provides less disruption of quality training prior to competition.

Approximately70%ofadaptationshavebeendemonstratedtooccurfollowingSTHA,evidencedby

reductionsinthermoregulatoryandcardiovascularstrain,combinedwithanimprovedsudomotor

function (Poirier et al., 2015).Acknowledgingpreviousobservations thatmales typically have a

superiorsudomotorfunctioncomparedwithfemales(GagnonandKenny,2011;Inoueetal.,2005),

itmaybeexpectedthatfemalesachievesuperiorsudomotoradaptationfollowingSTHAcompared

withmales.However,Sunderlandandcolleagues(2008)onlyachievedpartialHAintrainedfemales

witha33%increaseinintermittentsprintperformanceintheheat,despitenoalterationsinclassic

indicatorsofHAincludingHR,Tre,andSWR,followingSTHA.Thesetypicaladaptiveresponseshave

beenpreviouslyobserved intrainedmalesfollowingSTHA(Buonoetal.,1998;Fujiietal.,2012;

Garrettetal.,2011;Poirieretal.,2015;Racinaisetal.,2012);suggestingfemalesmayrequireLTHA

toachieveadaptation.Duetotheself-pacedexerciseadministeredpreandpostHA,participants

wereexercisingatahigherabsolute intensity followingHA,suggestingan increase inmetabolic

heatproduction;potentiallynegatinganyimprovementsinthermoregulationachievedthroughHA.

ResearchisrequiredtodeterminetheextenttowhichfemalesadapttoSTHAwhenusingafixed

intensityHTTtomonitoradaptations.

The primary aim of this study was to compare males and females’ thermoregulatory,

cardiovascular,andsudomotoradaptationtoSTHAandLTHAusingthecontrolledhyperthermia

modelofHA.Theprimaryhypothesis is thatmaleswillachievegreaterreductions inTreandHR

following STHA compared with females. The secondary hypothesis is that there will be no

differencesinthereductioninTreandHRbetweenmalesandfemalesfollowingLTHA.Thetertiary

hypothesis is that females will achieve a greater increase in SWR following STHA and LTHA

comparedwithmales.

121


Participants

Sixteen(8M,8F)athletes(3.2)(table6.1)providedwritteninformedconsenttoparticipateinthe

study,whichwasapprovedbytheUniversityofBrightonResearchEthics&GovernanceCommittee

and conducted in line with health and safety guidelines (3.1.) and in accordance with the

DeclarationofHelsinki,2013.


Notes:BM,bodymass;VnO2peak,peakoxygenconsumption.*denotessignificantdifferencebetweensexes

(p≤0.05).

Preliminarytesting


(3.5.)followedbyagradedexercisetest.AgradedexercisetestwasperformedtodetermineVnO2

peak(3.14.).Breath-by-breathexpiredmetabolicgaswasmeasuredusingonlinegasanalysis(3.9.)

andacapillarylactatesamplewastakenoncompletionofthetest(3.11.).Aregressionequation

wascomputedfromthedataobtainedtocalculatetherequiredintensity(65%VnO2peak)forthe


Males Females

Number 8 8

Age(yrs.) 22±6 20±1

Stature(m) 1.78±0.06 1.64±0.07*

BM(kg) 74.16±6.92 58.89±7.70*

Sumof4skinfolds(mm) 34±5 45±13*

AbsoluteVSO2peak(L.min-1) 3.63±0.69 2.69±0.30*

RelativeVSO2peak(mL.kg-1.min-1) 48.54±5.68 46.10±5.82

EndPoweroutput(W) 299±33 200±25*

122

Experimentaldesign


conducted in linewith themenstrual cycle standardisation guidelines (3.6.). Three participants

wereusingoralcontraceptive(Yasmin(30µgethinylestradiol/3mgdrospirenone).Alltrialswere

conducted in linewith pre-trial diet and exercise standardisation (3.4.). Participants provided a

urinesampleonarrivaltoensureadequatehydration(3.4.).Participantsperformed10HAsessions

(3.14.)separatedbythreeRHTT(3.16.).ThefirstRHTTwasperformed48-hrspriortobeginningHA

(RHTT1),thesecond48-hrsfollowing5-dHA(RHTT2)andthethird48-hrsfollowingtenHAsessions

(RHTT3). All experimental trials were performed in a hot environment (3.7.). Exercise was

terminatedinlinewithUniversityguidelines(3.1.).Therewerenoincidencesofearlytermination

ofexerciseduringtheRHTTwithinthisstudy.


TheRHTT(3.16.)wasperformedonthreeoccasions.Followinga20-minstabilisationperiodina

temperate environment (3.7.), measures of Tre, Tsk, (3.8.) and HR (3.9.) were recorded, and

participantsenteredtheenvironmentalchamber.ToweldriednudeBMwasrecordedtocalculate

SWR (3.12.). Throughout the test Tre, Tsk, and HR were recorded at 5-min intervals. Mean Tsk

(Equation2.4)wassubsequentlycalculated.

Heatacclimation

HAinvolvedtwoblocksoffiveconsecutivecontrolledhyperthermiasessionsseparatedby48-hrs

(3.14.).

Statisticalanalyses

AlldatawerefirstcheckedfornormalityusingShapiro-Wilkandcorrectedforsphericityusingthe

Greenhouse Geisser method. An independent samples t-test was used to identify differences

betweenmaleandfemalecharacteristics.Atwo-way(3x2)mixeddesignANOVAwasperformed

toidentifydifferencesbetweentheperformanceandphysiologicalcharacteristicsduringSTHAand

LTHA,thephysiologicalresponsesonD1,D5andD10ofHAandthephysiologicalresponsesduring

RHTT1,RHTT2andRHTT3 (3.17.4.).Whenamaineffector interactioneffectwas found, results

werefollowedupusingaBonferronicorrectedposthoccomparison(3.17.6.).Effectsizes(partial

Eta squared (np2)) were calculated to analyse the magnitude and trends of the interventions

123

(3.17.5.). All data was analysed using a standard statistical package (SPSS version 20.0), and

reportedasmean±SD.Statisticalsignificancewasacceptedatthelevelofp≤0.05.

6.4. Results

Performanceresponsesduringheatacclimation(D1-D5andD5-D10)

Table6.2presents themean±SDdata for theperformanceandphysiological responsesduring

STHAandLTHA.Allparticipantscompletedten,90-minHAsessions.ANOVArevealedamaineffect

ofHAphaseonexerciseduration(F (1,14)=7.728,p=0.015,np2=0.356).Exercisedurationwas

lower in STHA (70 ± 8min) comparedwith LTHA (75 ± 7min). Therewasno interaction effect

betweenHAphaseandsexforexerciseduration(F(1,14)=0.340,p=0.569,np2=0.024).

TherewasamaineffectofHAphaseonexerciseintensity(F(1,14)=4.710,p=0.048,np2=0.252).

ExerciseintensitywaslowerinSTHA(57±6%VnO2peak)comparedwithLTHA(59±5%VnO2peak).

TherewasnointeractioneffectbetweenHAphaseandsexforexerciseintensity(F(1,14)=0.587,p

=0.456,np2=0.04).

TherewasamaineffectofHAphaseontotalwork(F(1,14)=16.272,p<0.001,np2=0.538).Total

workwaslowerinSTHA(484±105kJ)comparedwithLTHA(570±124kJ).Therewasnointeraction

effectbetweenHAphaseandsexfortotalwork(F(1,14)=0.186,p=0.673,np2=0.013).

ANOVArevealedamaineffectofHAphaseondurationofTre≥38.5°C(F(1,14)=4.982,p=0.042,np2

=0.262).ThedurationofTre≥38.5°CwashigherinSTHA(49±8min)comparedwithLTHA(46±8

min).TherewasnointeractioneffectbetweenHAphaseandsexfordurationofTre≥38.5°C(F(1,14)

=0.513,p=0.486,np2=0.035).

124

Table6.2Performanceandphysiologicalresponsesduringshort-termheatacclimationandlong-termheatacclimation.Mean±SD.

STHA LTHA

Males Females Males Females

ExerciseDuration(min) 69±7 70±9 76±7* 74±7*

MeanExerciseIntensity(%VBO2peak) 55±7 59±5 58±6* 60±4*

TotalWorkDone(kJ) 562±70 413±79 653±116* 487±63*

DurationofTre>38.5°C(min) 48±9 51±7 45±8* 47±9*

Trerest(°C) 37.12±011 37.33±0.46 36.72±0.24* 37.00±0.25*

MeanTre(°C) 38.20±0.10 38.19±0.21 38.17±0.14 38.23±0.10

HRrest(beats.min-1) 74±3 80±5 62±6* 67±4*

MeanHR(beats.min-1) 150±9 151±9 151±9 154±8

SWRBSA(g.hr-1.m2) 276±65 278±53 317±65* 341±194*

Notes:LTHA,long-termheatacclimation;HR,heartrate;SWRBS,Sweatraterelativetobodysurfacearea;STHA,short-termheatacclimation;Tre,rectaltemperature.*denotessignificantly

differencetoSTHA(p≤0.05).

125

Physiologicalresponsesduringheatacclimation(D1-D5andD5-D10)

Thermoregulatoryresponses

TherewasamaineffectofHAdayonTrerest(F(2,28)=37.281,p≤0.001,np2=0.727).Therewasa

reductioninTrerestfromD1toD5(-0.26±0.19°C,p≤0.001),fromD5toD10(-0.21±0.28,p=

0.002),andfromD1toD10(-0.47±0.20,p≤0.001).TherewasnointeractioneffectbetweenHA

dayandsexonTrerest(F(2,28)=1.732,p=0.195,np2=0.110).

There was no main effect of HA phase on mean Tre (F (1, 14) = 0.000, p = 0.988, np2 = 0.000).

Furthermore,therewasnointeractioneffectbetweenHAphaseandsexonmeanTre(F(1,14)=0.872,

p=0.366,np2=0.059).

Cardiovascularresponses

TherewasamaineffectofHAdayonHRrest(F(2,28)=24.137,p≤0.001,np2=0.633).Therewere

nochangesinHRrestfromD1toD5(-4±6beats.min-1,p=0.070).TherewasareductioninHRrest

fromD5toD10(-6±4beats.min-1p≤0.001)andfromD1toD10(-10±7beats.min-1,p≤0.001).

TherewasnointeractioneffectbetweenHAdayandsexonHRrest(F(2,28)=2.117,p=0.139,np2

=0.131).

There was no main effect of HA phase on mean HR (F (1, 14) = 3.059, p = 0.102, np2 = 0.179).

Furthermore, therewasno interactioneffectbetweenHAphaseand sexonmeanHR (F (1,14) =

0.716,p=0.412,np2=0.049).

Sudomotorresponses

TherewasamaineffectofHAdayonSWRBSA(F(2,28)=16.266,p≤0.001,np2=0.537).Therewasan

increasefromD1toD5(89±144g.hr-1.m2,p=0.043),fromD5toD10(87±114g.hr-1.m2,p=0.014)

andfromD1toD10(177±134g.hr-1.m2,p≤0.001).TherewasnointeractioneffectbetweenHA

dayandsexonSWRBSA(F(2,28)=2.806,p=0.077,np2=0.167).

TherewasamaineffectofHAphaseonSWRBSA(F(1,14)=21.737,p≤0.001,np2=0.608).SWRBSAwas

lower in STHA (277 ± 58 g.hr-1.m2) compared with LTHA (329 ± 79 g.hr-1.m2). There was no

interactioneffectbetweenHAphaseandsexonSWRBSA(F(1,14)=0.987,p=0.337,np2=0.066).

126

Thermoregulatoryresponsetoshort-termandlong-termheatacclimation

Table6.3presentsthemean±SDdataformalesandfemalesphysiologicalresponsesduringRHTT1,

RHTT2,andRHTT3.TherewasamaineffectofRHTTforTrerest(F(2,28)=26.084,p≤0.001,np2=

0.651).TrerestreducedfollowingSTHA(RHTT1toRHTT2)(-0.13±0.16°C,p=0.002),LTHA(RHTT2toRHTT3)(-

0.13±0.16°C,p=0.006)andLTHA(RHTT1toRHTT3)(-0.26±0.16°C,p≤0.001).Therewasaninteraction

effectbetweenRHTTandsexforTrerest(F(2,28)=5.282,p=0.011,np2=0.274).Trerestreduced

followingSTHA(RHTT1toRHTT2)inmales(-0.24±0.16°C,p≤0.001),butnodifferenceswereobserved

infemales(-0.02±0.08°C,p=0.597).TrerestreducedfollowingLTHA(RHTT2toRHTT3)forfemales(-0.22

±0.12°C,p≤0.001),butnodifferenceswereobservedinmales(-0.04±0.15°C,p=0.459).Trerest

reducedfollowingLTHA(RHTT1toRHTT3)forbothmales(-0.28±0.17°C,p≤0.001)andfemales(-0.24±

0.17°C,p≤0.001).

TherewasamaineffectofRHTT forpeakTre (F (2,28)=17.972,p≤0.001,np2=0.532).PeakTre

reducedfollowingSTHA(RHTT1toRHTT2)(-0.23±0.32°C,p=0.018),LTHA(RHTT2toRHTT3)(-0.26±0.30°C,p

=0.008)andLTHA(RHTT1toRHTT3)(-0.46±0.36°C,p≤0.001).Therewasaninteractioneffectbetween

RHTTandsexforpeakTre(F(2,28)=3.339,p=0.050,np2=0.193).PeakTrereducedfollowingSTHA

(RHTT1toRHTT2)formales(-0.39±0.36°C,p≤0.001),butnodifferenceswereobservedinfemales(-0.07

±0.18,p=0.504).PeakTre reduced followingLTHA (RHTT2toRHTT3) for females (-0.41±0.24°C,p≤

0.001),butnodifferenceswereobserved inmales (-0.05±0.26°C,p=0.590).PeakTre reduced

followingLTHA(RHTT1toRHTT3)forbothmales(-0.44±0.45°C,p=0.005)andfemales(-0.48±0.27°C,p

=0.003).

Figure6.1presentsTredataat5-minintervalsduringtheRHTTforbothmalesandfemales.There

wasamaineffectofRHTTandtimeonTre(F(12,168)=2.343,p=0.008,np20.143).FollowingSTHA

(RHTT1toRHTT2)therewasareductioninTreat5(p≤0.001),10(p=0.008),15(p=0.027),20(p=0.007)

and25(p=0.018)min.FollowingLTHA(RHTT2toRHTT3)therewerenodifferencesinTreat5(p=0.265),

10(p=0.347),15(p=0.138),20(p=0.346)and25(p=113)min.FollowingLTHA(RHTT1toRHTT3)there

wasareductioninTreat5(p=0.002),10(p=0.005),15(p=0.006),20(p=0.002)and25(p≤0.001)

min.TherewasnointeractioneffectbetweenRHTTandtimeandsexforTre(F(12,168)=1.055p=

0.402,np2=0.070).

TherewasnomaineffectofRHTTforTrechange(F(2,28)=2.502,p=0.100,np2=0.152).Therewas

nointeractioneffectbetweenRHTTandsexobservedforTrechange(F(2,28)=0.513,p=0.604,np2

=0.035).

127

Table6.3Physiologicalvariablesduringbaselinetesting(RHTT1),followingSTHA(RHTT2)andfollowingLTHA(RHTT3).Mean±SD.

RHTT1 RHTT2 RHTT3

Males Females Males Females Males Females

Trerest(°C) 37.16±0.11 37.23±0.29 36.92±0.19* 37.21±0.34 36.88±0.18* 36.94±0.29*†

Trepeak(°C) 38.67±0.25 39.18±0.39 38.28±0.24* 39.12±0.44 38.24±0.40* 38.71±0.42*†

Trechange(°C) 1.51±0.26 1.95±0.31 1.37±0.25 1.91±0.27 1.35±0.41 1.72±0.38

Tskpeak(°C)37.30±0.53 37.66±0.95 36.80±0.36 37.25±0.55 36.27±0.77 36.58±0.72*†

HRrest(beats.min-1) 71±9 78±10 66±8 71±7 63±4 62±7

HRpeak(beats.min-1) 185±11 190±11 170±12* 185±11 172±10* 180±11*

SWR(g.hr-1) 1610±435 524±244 1588±359 1215±334* 2170±609*† 1300±353*

SWRBSA(g.hr-1.m2) 838±215 326±156 827±168 754±260* 1135±324*† 798±229*

Notes:HR,heart rate;RHTT, runningheat tolerancetest;SWR,Sweatrate;SWRBSA,Sweatraterelativetobodysurfacearea;Tre, rectal temperature;Tsk, skin temperature.*denotes

significantdifferencetoRHTT1(p≤0.05).†denotessignificantdifferencetoRHTT2(p≤0.05).

128

TherewasamaineffectofRHTT forpeakTsk (F (2,28)=19.085,p≤0.001,np2=0.577).PeakTsk

reducedfollowingSTHA(RHTT1toRHTT2)(-0.45±0.62°C;p=0.038),LTHA(RHTT2toRHTT3)(-0.60±0.62°C,p

=0.007)andLTHA(RHTT1toRHTT3)(-1.05±0.74°C,p≤0.001).Therewasnointeractioneffectbetween

RHTTandsexobservedforpeakTsk(F(2,28)=0.088,p=0.916,np2=0.006).

Figure6.1Rectal temperature (Tre)at5-min intervalsduring therunningheat tolerance test for

males(left)andfemales(right).GreymarkersrepresenttheRHTT1,blackmarkersRHTT2andwhite

markerRHTT3.Mean±SD.N=16(8M,8F).

*denotessignificantdifferenceinSTHA(RHTT1toRHTT2)(p≤0.05).+denotessignificantdifferenceinLTHA(RHTT2

toRHTT3)(p≤0.05).†denotessignificantdifferenceinLTHA(RHTT1toRHTT3)(p≤0.05).

Cardiovascularresponsetoshort-termandlong-termheatacclimation

TherewasamaineffectofRHTTforHRrest(F(2,28)=11.177,p≤0.001,np2=0.444).FollowingSTHA

(RHTT1toRHTT2)therewerenoobserveddifferencesinHRrest(-6±11beats.min-1,p=0.117).HRrest

reducedfollowingLTHA(RHTT2toRHTT3)(-6±8beats.min-1,p=0.027)andLTHA(RHTT1toRHTT3)(-12±12

36.6

37.0

37.4

37.8

38.2

38.6

39.0

39.4

39.8

0 5 10 15 20 25 30

T re(°C

)

Time(min)

*+Males *+

0 5 10 15 20 25 30

Time(min)

RHTT1

RHTT2

RHTT3

+† Females +†

129

beats.min-1,p=0.003).TherewasnointeractioneffectbetweenRHTTandsexobservedforHRrest

(F(2,28)=0.942,p=0.402,np2=0.063).

TherewasamaineffectofRHTTforpeakHR(F (2,28)=19.916,p≤0.001,np2=0.587).PeakHR

reducedfollowingSTHA(RHTT1toRHTT2)(-9±10beats.min-1,p=0.002),showednodifferencesfollowing

LTHA(RHTT2toRHTT3)(-2±8beats.min-1,p=1.000),butreducedfollowingLTHA(RHTT1toRHTT3)(-11±6

beats.min-1,p≤0.001).TherewasaninteractioneffectbetweenRHTTandsexforpeakHR(F(2,28)

=3.598,p=0.041,np2=0.204).PeakHRreducedfollowingSTHA(RHTT1toRHTT2) inmales(-14±12

beats.min-1,p≤0.001),nodifferenceswereobserved in females (-5±3beats.min

-1,p=0.164).

TherewerenodifferencesobservedforpeakHRfollowingLTHA(RHTT2toRHTT3)inbothmales(2±10

beats.min-1,p=0.505)andfemales(-5±5beats.min

-1,p=0.076).PeakHRreducedfollowingLTHA

(RHTT1toRHTT3) forbothmales(-13±7beats.min-1,p≤0.001)andfemales(-10±6beats.min

-1,p≤

0.001).

Figure6.2presentstheHR5-minintervaldataforbothmalesandfemales.Therewasnomaineffect

ofRHTTandtimeonHR(F(12,168)=0.845,p=0.604,np2=0.057).Therewasnointeractioneffect

betweenRHTTandtimeandsexforHR(F(12,168)=1.055p=0.401,np2=0.070).

Sudomotorresponsetoshort-termandlong-termheatacclimation

TherewasamaineffectofRHTTforSWR(F(2,28)=12.207,p≤0.001,np2=0.466).SWRincreased

followingSTHA(RHTT1toRHTT2)(334±590g.hr-1,p=0.042).Therewerenodifferencesobservedfor

LTHA(RHTT2toRHTT3)forSWR(334±636g.hr-1,p=0.131)however,anincreasewasobservedfollowing

LTHA(RHTT1toRHTT3)(668±529g.hr-1,p≤0.001).TherewasaninteractioneffectbetweenRHTTand

sexforSWR(F(2,28)=3.661,p=0.039,np2=0.270).SWRincreasedfollowingSTHA(RHTT1toRHTT2)in

females(691±412g.hr-1,p≤0.001),butnodifferenceswereobservedinmales(-22±533g.hr,p

=0.896).SWRincreasedfollowingLTHA(RHTT2toRHTT3)formales(583±638g.hr-1,p=0.016),butno

differenceswereobservedinfemales(85±564g.hr-1,p=0.696).SWRincreasedfollowingLTHA

(RHTT1toRHTT3)forbothmales(560±594g.hr-1,p≤0.001)andfemales(776±470g.hr

-1,p≤0.001).

130

Figure6.2Heartrate(HR)at5-minintervalsduringtherunningheattolerancetestformales(left)

andfemales (right).Greymarkersrepresent theRHTT1,blackmarkersRHTT2andwhitemarker

RHTT3.Mean±SD.N=16(8M,8F).

*denotessignificantdifferenceinSTHA(RHTT1toRHTT2)(p≤0.05).†denotessignificantdifferenceinLTHA(RHTT1

toRHTT3)(p≤0.05).

Therewas amain effect of RHTT for SWRBSA (F (2, 28) = 11.947,p ≤ 0.001,np2 = 0.460). SWRBSA

increasedfollowingSTHA(RHTT1toRHTT2)(334±590g.hr-1.m

2;p=0.029).Therewerenodifferences

observedforLTHA(RHTT2toRHTT3)(334±636g.hr-1.m

2,p=0.210),however,anincreasewasobserved

followingLTHA(RHTT1toRHTT3)(668±529g.hr-1.m

2,p≤0.001).Therewasaninteractioneffectbetween

RHTTandsexonSWRBSA(F(2,28)=3.939,p=0.031,np2=0.220).SWRBSAincreasedfollowingSTHA

(RHTT1toRHTT2)forfemales(428±269g.hr-1.m

2,p≤0.001),butnodifferenceswereobservedinmales

(-11±286g.hr-1.m

2,p=0.909).SWRBSAincreasedfollowingLTHA(RHTT2toRHTT3)formales(308±346

g.hr-1.m

2,p=0.029),butnodifferenceswereobservedinfemales(44±373g.hr

-1.m

2,p=0.733).

SWRBSAincreasedfollowingLTHA(RHTT1toRHTT3)forbothmales(297±314g.hr-1.m

2,p=0.015)and

females(472±291g.hr-1.m

2,p≤0.001).

50

70

90

110

130

150

170

190

210

230

0 5 10 15 20 25 30

HR(be

ats.min¯¹)

Time(min)

Males *†

0 5 10 15 20 25 30

Time(min)

RHTT1

RHTT2

RHTT3

Females †

131

6.5. Discussion

The understanding of HA on experimentation using humans is primarily based upon male

participants. The application of HA to femalesmay be inappropriate due to sex differences in

thermoregulation.ThisstudyexaminedthesexdifferencesinthetemporalpatterningtoSTHAand

LTHA.Thedatademonstrates thatbothmalesand femalesachievepartialadaptation following

STHA;withmalesdemonstratinga reduction in thermoregulatoryandcardiovascular strainand

femalesdemonstratinganincreasedsudomotorfunction.FollowingLTHA,bothmalesandfemales

achievedadditionaladaptation;withfemalesdemonstratingareductioninthermoregulatorystrain

andmalesan increasedsudomotorfunction.Theseresultssuggestthatbothmalesandfemales

respondtoSTHAhowever,femalesrequireLTHAtoestablishreductionsinthermoregulatoryand

cardiovascularstrain.Thesefindingssupporttheaimofthestudywiththeprimary,secondaryand

tertiaryhypothesisalsobeingaccepted.

Short-termheatacclimation

Following STHA, approximately 70% ofmaximal adaptations has been reported to be achieved

(Poirieretal.,2015). Inthecurrentstudy,malesdemonstratedmoreadaptationfollowingSTHA

comparedwithfemales,withareductioninTrerest(-0.24±0.16°C)andpeakTre(-0.32±0.36°C);

thesechangesdidnotalterTrechange.ThemagnitudeofreductioninTreisverysimilartothe0.3°C

observedbyGarrettandcolleagues(2012)following5-dcontrolledhyperthermiaHA.Endurance

performanceismarkedlyimpairedinhotcomparedtotemperateenvironmentduetoanincrease

inTccausingadecreaseincentralactivation(NyboandNielsen,2001).AttenuationofthepeakTre

may lessen or delay the likelihood of individuals obtaining or expressing signs of heat-related

illnesseswhentraining,workingorcompetingintheheat,demonstratingtheeffectivenessofSTHA

inmales.

In thecurrentstudy,exercise intensity,exerciseduration,andtotalworkperformedwashigher

duringLTHAcomparedwithSTHA.Thisincreasedexerciseintensitywasadministeredtoelicitand

maintainthetargetTcof38.5°C.ThehigherexerciseintensityduringLTHAwouldresultinahigher

metabolic heat production compared with STHA. Since total heat loss during exercise is

predominantlyafunctionofevaporativeheatloss,agreaterrateofmetabolicheatproductionin

LTHA,withcomparableTrevaluesachieved,suggestsanincreaseinevaporativeheatlossandthus

reduced heat storage. These findings support that partial heat adaptationwas achieved during

STHA.

132

AreductionincardiovascularstrainwasachievedfollowingSTHAinmaleparticipants,evidenced

bya reduction inpeakHR. The14±12beats.min-1reduction inpeakHR is in accordancewith

previousobservationsfollowingSTHAusingcontrolledhyperthermia(Garrettetal.,2012;Patterson

et al., 2004a). The reduction in cardiovascular strain is potentially due to an increase in blood

volume,preservingstrokevolumeandreducingheattransferfromthebody’scoretotheskinand

ultimately,totheexternalenvironment.

Sunderland and colleagues (2008) assessed the effect of 4-d HA on female games players’

intermittent sprintperformance in theheat. Intermittent sprintperformance increasedby33%,

following 4-d HA, however there were no differences in peak Tre, peak HRand SWR. The self-

regulatednatureof the intermittent sprintprotocolusedprovidesnostandardisedendogenous

thermal load, potentially constraining adaptation in some individuals (Taylor andCotter, 2006).

Furthermore,anyreductioninthermoregulatoryandcardiovascularstrainmayhavebeennegated

duetoparticipantsperformingmoreworkfollowingHA.Thefindings inthecurrentstudyarein

agreementwiththesepreviousreports,withnoreductionsincardiovascularandthermoregulatory

strainduringSTHAinfemaleparticipants.

IncreasedSWRhavebeenreportedtooccurfollowingSTHA(Buonoetal.,2009;Pattersonetal.,

2004;Poirieretal.,2015).Pattersonandcolleagues(2004a)reportedthatonlysixHAsessionswere

requiredtoelicitpartialsudomotoradaptation,evidencedbyanelevatedlocalSWR.Thefindings

inthecurrentstudysupportpreviousfindingswitha428±269g.hr-1.m

2increaseinSWRinfemale

participantsfollowingSTHA.Although,femalesinthecurrentstudyhadalowerstimulusforsweat

production,duetoexercisingatalowermetabolicheatproductionpotentially,theynonetheless

improvedsweatproductiontoagreaterextentcomparedtomales,particularly followingSTHA.

Thisissurprising,basedonpreviousfindingswhichsuggestthattheproductionofsweatisthemain

driverforimprovementsinsweatingduringHA(Buonoetal.,2009).Assuch,itcouldbearguedthat

femalesadaptduringSTHAtoagreaterextentthanmales.However,theobservedincreaseinSWR

didnotresult inareducedthermalstraininfemaleparticipants.NoplateauwasobservedinTre

duringtheRHTTinfemaleparticipants,thustheenhancedSWRdidnotoffsettheuncompensatable

rateofmetabolicheatproductionobserved.

AnincreaseinwholebodySWRobservedinfemalesfollowingSTHAinthecurrentstudy,suggests

eitheranalteredafferentneuralactivityfromtheperipheralorcentralthermo-receptorscausing

differentintegrationofthermalinformation,analteredefferentneuralactivityforagivenlevelof

afferentinput,oranalteredeffectorresponse.Sexmodulatesperipheralcontrolofthesudomotor

function,thisisevidencedbyareducedthermosensitivity;resultinginfemaleshavingareduced

133

SWRcomparedwithmales(GagnonandKenny,2011).Consequently,itmaybehypothesisedthat

theenhancedsudomotorfunctioninthefemaleparticipantsfollowingSTHAinthecurrentstudyis

asaresultofperipheralchangestothethermosensitivityoftheeccrinesweatglands.Thepotential

mechanisms for this include an increased cholinergic sensitivity of the eccrine sweat gland and

increaseglandularhypertrophy(Buonoetal.,2009;LorenzoandMinson,2010).

In the current study, therewerenoobservedchanges inSWRduringSTHA (RHTT1 toRHTT2) inmale

participants. These findings may be due to a lower peak Tre in RHTT2 compared with RHTT1.

Specifically,inmaleparticipantsthepeakTreinRHTT1was38.67±0.25°CwhichproducedaSWRBSA

of838±215g.hr-1.m

2.ThepeakTreinRHTT2was38.28±0.24°CwhichproducedaSWRBSAof827±

168g.hr-1.m

2.ItiswellreportedthatanincreaseinTcstimulatessudomotorfunctionduringexercise

intheheat(Sawkaetal.,1989).Recentfindings,suggestthatwholebodySWRmayunderestimate

thetrueadaptationthatoccurredtothesweatglandfunctionfollowingHA(Buonoetal.,2009a;

Inoueetal.,1999).Buonoetal.(2009)reported20%increaseinwholebodySWR,whilepilocarpine

induced SWR increases by 63% following 8-dHA. Furthermore, Inoue et al. (1999) reportedno

changes in whole body SWR, while a significant improvement was observed for methycholine

inducedSWRfollowing8-dHA.

Long-termheatacclimation

The adaptive effects of LTHA arewell established, such that the extent towhich an individual

physiologicallyadaptstoHAisdependentuponthelengthofexposuretoheatstressconditions.In

the current study, therewasa reduction in the combined thermoregulatory and cardiovascular

strainandanenhancedsudomotorfunctionfollowingLTHAinallparticipants.Thesefindingsarein

accordance with the results reported by Avellini and colleagues (1980) when assessing sex

differencesinadaptationto10-dfixedintensityHA.Malesandfemaleswerereportedtoexpressa

similar adaptive response, evidencedby a reduction in Tre, an increased SWRand an improved

exercise capacity. Participants worked at the same absolute exercise intensity during the HA

session,therefore,theremayhavebeenvarietyinthephysiologicalstrainplaceduponparticipants.

When work rate remains constant, thermal strain during sequential exposures progressively

declines,constrainingadaptation(TaylorandCotter,2006).

Since controlled hyperthermia HA ensures equal thermal strain during every session, it can be

assumed that adaptation was not constrained during sequential sessions in the current study,

establishingmore complete adaptation (Taylor and Cotter, 2006). Additional adaptations were

observed during LTHA for the female participants, evidenced by reductions in measures of

134

thermoregulatory and cardiovascular strain from RHTT2 to RHTT3. These observed differences

werenotpresent in themaleparticipants, thus females requireLTHAtoestablish reductions in

thermoregulatoryandcardiovascularstrain.

LTHA established an improved sudomotor response in the current study. Increased SWR is not

uniquetothecurrentstudywithHAknowntoimproveperipheralandcentralmechanismsinvolved

insudomotorfunction,viaenhancedsweatglandsensitivity(Buonoetal.,2009a)andreductions

in the onset threshold for sweating, enhancing evaporative heat loss (Yamazaki andHamasaki,

2003).Completesudomotoradaptationhasbeensuggestedtotakebetween10-14dtoestablish

(ArmstrongandMaresh,1991),butthisbiphasicadaptationmaybemoreprotocoldependent.In

thecurrentstudyfemalesobtainednoadditionalbenefittothesudomotorfunctionasaresultof

LTHA,however,anincreaseinSWRwasobservedfromRHTT2toRHTT3inmaleparticipants.

6.6. Limitations

TheexerciseelicitedduringtheHAsessionswasperformedat65%ofVhO2peak.Females inthe

currentstudyhadalowerabsoluteVhO2peakcomparedwiththemaleparticipants,consequently

theyworkedatalowermetabolicheatproductionprovidingalowerstimulusforsweatproduction

(CramerandJay,2014;Gagnonetal.,2013,2008).Furthermore,femalesinthecurrentstudyhad

alowerBMcomparedwiththemaleparticipantswhichentailslessheatstorage,andthereforea

lower exercise intensity is required to increase their Tc to 38.5°C (Gagnon et al., 2009).

Consequently,thestressimposedduringtheHAsessionswaslikelylowerinfemaleparticipants,

potentially constraining adaptation and influencing the time course of adaptation, due to

inadequate endogenous heat strain. Future research is warranted to quantify these potential

differences between males and females. Future work should involve the implementation of a

controlled hyperthermia HA protocol where workload is administered using relative heat

production.ThismayfurtheroptimiseadaptationtoHAbyreducingindividualvariabilityassociated

withmetabolicheatproduction(Cramer&Jay2014)

Future work should implement greater control over hormonal alterations which alter

thermoregulatory responses associated with the menstrual cycle between repeated trials. An

elevationinprogesteroneconcentration,associatedwiththelutealphaseofthemenstrualcycle,

alters resting Tc, the threshold for sweating, and cutaneous vasodilation and consequently,

tolerancetoexerciseheatstress(Inoueetal.,2005).Inthecurrentstudy,theminimumnumberof

daystheprotocolrequiredforcompletionwas16,thuscrossingovertwomenstrualcyclephases.

Forthoseparticipantsnotusingoralcontraception,RHTT1andRHTT2wereperformedduringthe

135

follicularphaseoftheirself-reportedmenses,whenrestingTcandthethresholdfortheonsetof

sweatingandcutaneousvasodilation is lowercomparedwith the lutealphase.However,RHTT3

wasperformedduringthelutealphaseoftheirself-reportedmenses.Consequently,theextentof

the adaptation reported in females may have been smaller due to alterations in hormone

concentrationsofprogesteroneassociatedwiththemenstrualcycle(Inoueetal.,2005).Controlling

forthehormonalalterationassociatedwiththemenstrualcyclethroughoutHAwouldprovidea

greaterunderstandingintothetrueadaptationpresent.

Furthermore,inthecurrentstudy,changesinPVandfluidregulationwerenotmeasured,bothof

whichassistinthemaintenanceofanelevatedSWRandareducedcardiovascularstrainassociated

withHA(Taylor,2014).Consequently,theeffectoftheseadaptationsontheimprovementinSWR

andreductionincardiovascularstraininthecurrentstudyisnotknown.Futureresearchshouldbe

conductedobservingthesevariableswhilstaccountingforhormonalfluctuationsassociatedwith

themenstrualcycle,duetotheirknowneffectonfluidbalanceandPV.

6.7. Conclusion

Inthecurrentstudy,HAwaseffectiveinattenuatingphysiologicalstrainandimprovingexercise-

heattoleranceinbothmalesandfemalesandthusmayreducethelikelihoodofobtainingaheat-

relatedillnessduringtrainingorcompetitionintheheat.STHAisapreferredregimeforathletes

sinceitiseasiertoadoptwhensustainingqualitytrainingandtaperingperformanceintheweeks

beforecompetition.ThesefindingssuggestthatwhilstSTHAmaybeeffectiveinachievingpartial

adaptation inmalesandfemales,femalesrequireLTHAtoestablishreductions incardiovascular

andthermoregulatorystrain.

ThesefindingsprovideoriginaldatawhichwillsupporttheimplementationofHAprotocolsthat

are tailored to target sex differences in the temporal patterning of adaptation. An additional

elementofHAisanacquiredcellularthermotolerance.Study4(Chapter7)willcomparemalesand

females’cellularadaptation,specificallyHsp72mRNAacrossthecourseofSTHAandLTHA.

136

7. SexcomparisonofleukocyteHsp72mRNAtranscriptionduringheatacclimation

________________________________________________________________________________

InStudy3(Chapter6)itbecameapparentthatfemales’phenotypicresponsestoHAdifferedfrom

thoseofmales.AnadditionalelementtoHAinanacquiredcellularthermotolerance.Thisstudywill

explorethedifferencesbetweenmalesandfemales’cellularadaptationtoSTHAandLTHAusing10

outofthe16participantspresentedinStudy3(Chapter6).

7.1. Abstract

Thermotolerance is an acquired state of increased cytoprotection achieved following single or

repeatedexposurestoheatstress,inpartcharacterisedbychangesintheintracellular72kdaheat

shockprotein(HSP72;HSPA1A).FemaleshavedemonstratedreducedexerciseinducedHSP72in

comparisontomales.ThisstudyexaminedsexdifferencesinHsp72mRNAtranscriptionduringheat

acclimation(HA)toidentifywhethersexdifferenceswerearesultofdifferentialgenetranscription.

Tenparticipants(5M,5F)performed10,90-mincontrolledhyperthermiaHAsessionsover12-d.

Leukocyte Hsp72 mRNA was measured pre and post D1, D5, and D10, via RT-QPCR. HA was

evidenced by a reduction in resting Tre (-0.36 ± 0.53°C) and resting heart rate [(HR); -13 ± 7

beats.min-1] following HA (p ≤ 0.05). During HA no difference (p > 0.05) was observed in ΔTre

betweenmales(D1=1.52±0.19°C;D5=1.56±0.38°C;D10=1.80±0.28°C)andfemales(D1=1.52

±0.47°C;D5=1.40±0.22°C;D10=1.80±0.28°C).ThiswasalsotrueofmeanTredemonstrating

equalityofthermalstimuliformRNAtranscriptionandHA.Therewerenodifferences(p>0.05)in

Hsp72mRNAexpressionbetweenHAsessionsorbetweenmales(D1=+1.8±1.5fold;D5=+2.0±

1.0fold;D10=+1.1±0.4fold)andfemales(D1=+2.6±1.8fold;D5=+1.8±1.4fold;D10=+0.9±

1.9fold).ThisexperimentdemonstratesthatthereisnodifferenceinHsp72mRNAincreasesduring

HA between sexes when controlled hyperthermia HA is utilised. Gender specific differences in

exercise-inducedHSP72reportedelsewherelikelyresultfrompost-transcriptionalevents.

7.2. Introduction

Repeatedexposuretostressfulthermalenvironmentsinitiatesheatadaptationinhumans(Taylor,

2014).Heatadaptationincorporatestheinterrelatedacclimationandthermotolerance(Horowitz,

2014;Sawkaetal.,2011a).Aheatacclimatedphenotypedescribesenhancedheat losseffector

responsesandhypervolemiawhichmitigatephysiological,perceptual,andfunctionaldetriments

137

to heat exposure (Périard et al., 2015; Taylor, 2014). Thermotolerance or acquired cellular

thermotoleranceisthenomenclatureusedtodescribecellularadaptationscausedbyasingle,or

repeatedsevere,butnon-lethalheatexposure[e.g.heatacclimation(HA)](Moseley,1997).

HArepeatedlyinitiatestheheatshockresponse(HSR),typicallyincreasingvariousbasalheatshock

proteins(HSP),includingHSP72(HSP72).Inresponseto10-dHA,baselineintracellularHSP72has

beenshowntoincreaseby18%(McClungetal.,2008)whilstHsp72mRNAdemonstratesapattern

wherebytranscriptionoccurswithineachHAsession(+195%)beforereturningtobaseline24-hrs

later(Gibsonetal.,2015a,2015c).ThesetransientHAmediatedcellularadaptationstoiHSP72can

confercytoprotectiontosubsequentthermal(McClungetal.,2008)andnon-thermal(Gibsonetal.,

2015c)stressorsinvitro(McClungetal.,2008)andinvivo(Leeetal.,2016).Eloquentin-vitrodata

demonstrates that cytoprotection to stress (thermal or otherwise) is abolished when HSP72 is

knockedout(Drewetal.,2014;Leeetal.,2004;Senfetal.,2013)orblocked(Kuennenetal.,2011).

HA mediated in-vivo cytoprotection is dependent upon sufficient Hsp72 mRNA transcription

(Moranetal.,2006)andsubsequentHSP72proteintranslation(SilverandNoble,2012).

ControlledhyperthermiaHAresultsinagreaterHsp72mRNAcomparedwithmatchedtrainingin

coolconditionsasaresultofgreaterendogenousstimulifortranscription(Gibsonetal.,2015c).

Due to the consistent endogenous thermal stimulus there is an equality of Hsp72 mRNA

transcription during 10-d controlled hyperthermia HA (Gibson et al., 2015a). Thus, controlled

hyperthermiaisapreferredHAmethodcomparedwithtraditionalexogenouslyprescribedHAsince

it induces robust Hsp72 mRNA responses, ensuring sufficient and consistent increases in

endogenous stimuli throughout an in vivo chronic intervention, particularly when comparing

independentgroups.

Mortonandcolleagues (2009) reporteda sex specificHSPadaptation inhumanskeletalmuscle

following6-wks.ofcontinuousandintervaltraining.Specifically,HSP70increasedby38±41%and

23±36%followingcontinuousandintervaltrainingrespectivelyinmales(n=5);howeverfemales

(n=5)hadnochanges(3±37%and4±14%increaserespectively),despitesimilartrainingstatus,

training prescription and training adaptations (VhO2max) (Morton et al., 2009). Differential sex

responses reported by Morton et al. (2009) may be attributed to cytoprotective effects of

oestrogen.Elevatedoestrogenhasbeenshowntoaffordcellularprotection(Shinoharaetal.,2004),

accordinglyincreasedoestrogeninfemalesversusmalesmayprovideamechanismforinhibited

changesinHSP72expression(Bombardieretal.,2009).Oestrogenbindstotheoestrogenreceptor,

whichisamemberofthesteroidfamilyofnuclearreceptorsandistheoestrogenresponseelement

intargetgenes,leadingtothetranscriptionalregulationofmanygenes(Ogitaetal.,2003).Gillum

138

etal.(2013)reportedhigherintracellularHSP72concentrationsfollowingasingleboutofexercise

intheheatinmalescomparedwithfemales(inboththefollicularandlutealphaseofthemenstrual

cycle), despite similar baseline values and identical endogenous stimuli for Hsp72 mRNA

transcription(Gillumetal.,2013).Differentialsexresponseswerealsosuggestedtobearesultof

oestrogenprovidingcellularprotectionandthus,decreasingthenecessityfortranslationofHSP72

infemales.Although,stress-mediatedsexspecificdifferencesintheHSP72havebeenseen(Gillum

etal.,2013;Mortonetal.,2009),theyhavenotbeenexaminedatanmRNAlevelacrossthecourse

ofcontrolledhyperthermiaHA.

DeterminationofHsp72mRNAtranscriptioninfemaleswouldfacilitateidentificationofwhether

theinhibitedHSP72responseresultedfromabsentgenesignalling,ormitigatedproteintranslation,

potentiallyduetoelevatedoestrogen(Gillumetal.,2013;Mortonetal.,2009).Absenceofdatain

femalepopulationscouldbeproblematicforpractitionerswhomayadoptHAprotocolsthatare

informedbymechanisticcellularadaptationsfrommaleonlycohorts(Gibsonetal.,2015a;Meeet

al., 2015). Thismay reduce themagnitude to which females are protected against heat injury

(Moranetal.,2006).

The aim of the current study was to determine whether the Hsp72 mRNA response during

controlledhyperthermiaHA,differedbetweenmalesand females. Itwashypothesised that the

Hsp72mRNAresponsewillbelowerinfemalescomparedtomalesacrossthecourseofcontrolled

hyperthermiaHA.


Participants

Based ona priori power analyses (3.17.1), four participants in each groupwould result in 95%

probabilityofdetectingadifferenceinHsp72mRNAacrossthecourseofcontrolledhyperthermia

HA(Gibsonetal.,2015a).Inlinewithpoweranalysis,andpreviousworkinthearea(Mortonetal.,

2009),ten(5M,5F)athletes(3.2)(table7.1)providedwritteninformedconsenttoparticipate.This

datawascollectedalongsideStudy3(Chapter6),howeverparticipantswerepre-determinedprior

todatacollectionfortheirinvolvementinbothstudies.ThestudywasapprovedbytheUniversity

ofBrightonResearchEthics&GovernanceCommitteeandconductedinlinewithhealthandsafety

guidelines(3.1)andinaccordancewiththeDeclarationofHelsinki,2013.

139


Age

(yrs.)

Stature

(m)

BM

(kg)

RelativeVQO2peak

(mL.kg-1.min-1)

Males(N=5) 24±7 1.75±0.03 70.1±5.1 46±4

Females(N=5) 20±1 1.63±0.09 57.1±4.9 46±4

Notes:BM,bodymass;VhO2peak,peakoxygenuptake

Preliminarytesting


(3.5.)followedbyagradedexercisetest.AgradedexercisetestwasperformedtodetermineVhO2

peak(3.14.).Breath-by-breathexpiredmetabolicgaswasmeasuredusingonlinegasanalysis(3.9.)

andacapillarylactatesamplewastakenoncompletionofthetest(3.11.).Aregressionequation

wascomputed for thedataobtained tocalculate the required intensity (65%VhO2peak) for the


Experimentaldesign


conductedinlinewiththemenstrualcyclestandardisationguidelines(3.6.).Twoparticipantwere

taking oral contraceptive: Yasmin (30 µg ethinylestradiol/ 3mg drospirenone). All trials were

conductedin linewithpre-trialdietandexercisestandardisation(3.4.). Inaddition,confounding

variablesincludingsmoking,caffeine,glutamine,alcohol,genericsupplementation,priorthermal,

hypoxic,andhyperbaricexposureswereallcontrolledinlinewithpreviousworkinthefield(Taylor

etal.,2011).Participantsprovidedaurinesampleuponarrivaltoensureadequatehydration(3.4.).

VolunteersperformedtwoblocksoffiveconsecutivedaysofcontrolledhyperthermiaHA(3.14.)in

a hot environment (3.7.) separated by 48 hr. Exercise was terminated in line with university

guidelines (3.1.). There were no incidences of early termination of exercise within this study.

Followinga20-minstabilisationperiod,measuresofTre (3.8.)andHR (3.9.)were recorded,and

140

participants entered the environmental chamber. Towel dried nude BM was also recorded to

calculateSWR(3.12.).ThroughouttheHAsessionsTre,HR,wererecordedat5-minintervals.

Blood sampling, RNA extraction, and one-step reverse transcription quantitative polymerase

chainreaction(RT-qPCR)

Inlinewithpreviousworkinthefield(Gibsonetal.,2015;Tuttleetal.,2015),venousbloodsamples

weretakenimmediatelybeforeandimmediatelypostexerciseheatexposureonD1,D5andD10

ofcontrolledhyperthermiaHA.Allbloodsamplesweredrawnfromtheantecubitalveininto6mL

EDTAVacuettetubes(GrenierBIO-One,Stonehouse,UK).1mLofvenousbloodwaspipettedinto

10mLof1in10redbloodcell lysissolution(10XredbloodCellLysisSolution;MiltenyiBiotech,

Bisley, UK). Samples were incubated for 15-min at room temperature then isolated via

centrifugationat400gfor5-minandwashedtwicein2mLphosphate-bufferedsalineat400gfor

5-min to isolateall leukocytes. Sampleswere then stored ina -83°C freezerand transported to

Bedfordshire University for the remainder of the analysis which was performed by a trained

laboratorytechnician.RNAwasextractedviathepreviouslyvalidatedacidguanidiumthiocyanate–

phenol–chloroform extraction method (Chomczynski and Sacchi, 1987). A standard RNA

concentrationwasproducedprior toPCrbyaddinga fixedamountofRNAtocellculturegrade

water togiveanequaldilution.Quantitywasdeterminedatanopticaldensityof260nmwhile

qualitywasdeterminedviathe260/280and260/230ratiosusingananodropspectrophotometer

(NanoDrop2000c;ThermoScientific,Waltham,MA,USA).

Hsp72mRNAwasquantifiedusingRT-QPCR.Primers(table7.2)weredesignedusingprimerdesign

software(PrimerQuestandOligoanalyzer;IntegratedDNATechnologies,Coralville,IA,USA).20μL

reactions containing 10 μL SYBR-Green RT-PCR Mastermix (Quantifast SYBRgreen Kit; Qiagen,

Manchester,UK),0.15μLforwardprimer,0.15μLreverseprimer,0.2μLreversetranscriptionmix

(QuantifastRTMix;Qiagen)and9.5μLsample(70ngRNA/μL)werepreparedinseparatetubes.

EachPCRreaction(RotorgeneQ;Qiagen)wasthenperformedas follows:10-min,50°C(reverse

transcription),5-min95°C(transcriptase inactivationandinitialdenaturation); followedby:10-s,

95°C(denaturation),30-s,60°C(annealingandextension)for40cycles.Fluorescencewasmeasured

following each cycle as a result of the incorporation of SYBR green dye into the amplified PCR

product.Melt curves (50 to95°C; rampprotocol5-s stages)wereanalysed foreach reaction to

ensure only the single gene of interest was amplified. The relative quantification of mRNA

expression foreachsamplewasassessedbydetermining the ratiobetween thecycle threshold

valueofthetargetmRNAandthecyclethresholdvaluesforβ2-microglobuinfoldchangeinrelative

mRNAexpressionwascalculatedusingthe2-ΔΔCTmethod(SchmittgenandLivak,2008).

141

Table7.2Hsp72mRNAprimersequences.

Gene NCBIAccession# Primer Sequence(5’→3’)Amplitude

length

Β2-Microglobulin

(β2-M)NM_004048

Forward

Reverse

CCGTGTGAACCATGTGACT

TGCGGCATCTTCAAACCT

91

Hsp72 NM_005345

Forward

Reverse

CGCAACGTGCTCATCTTTGA

TCGCTTGTTCTGGCTGATGT

198

Notes:NCBINationalCentreforBiotechnologyInformation

Statisticalanalysis

AlldatawerefirstcheckedfornormalityusingShapiro-Wilkandcorrectedforsphericityusingthe

GreenhouseGeissermethod.A twoway (3x2)mixeddesignanalysisofvariance (ANOVA)was

performed todeterminedifferencesbetween thephysiological andperformance characteristics

betweenD1,D5andD10inmalesandfemales(3.17.4.).Athree-way(3x2x2)mixeddesignANOVA

wasperformedtoidentifydifferencesbetweentheHsp72mRNA,preandpost,onD1,D5,andD10

of controlled hyperthermiaHAbetweenmales and females.When amain effect or interaction

effect was found, results were followed up using a Bonferroni corrected post hoc comparison

(3.17.6).Effectsizes[partialetasquared(np2)]werecalculatedtoanalysethemagnitudeandtrends

of the interventions (3.17.5.). All datawere analysed using a standard statistical package (SPSS

version20.0,IBM,Armonk,NewYork,USA)andreportedasmean±SD.Statisticalsignificancewas

acceptedatthelevelofp≤0.05.

7.4. Results

Evidenceofheatacclimation

Figure 7.1 presents the resting Tre and resting HR data for D1, D5 and D10 of controlled

hyperthermiaHA.TherewasamaineffectofdayonTrerest(F(2,16)=11.219,p≤0.001,np2=0.584).

NodifferenceswereobservedfromD1toD5(p=0.563).However,TrerestreducedfromD1toD10

(p=0.027)andfromD5toD10(p=0.003).Therewasnointeractioneffectbetweendayandsex

142

onTrerest(F(2,16)=3.287,p=0.064,np2=0.291).However,themeanreductioninTrerestfromD1

toD5was-0.28±0.21°Cinmaleswhereasinfemalestherewerenochanges(+0.08±0.24°C).The

meanreductioninTrerestfromD5toD10was-0.44±0.22°Cinfemaleswhereasinmalesthere

werenochanges(-0.09±0.06°C).

Figure7.1Restingrectaltemperature(Tre)(left)andrestingheartrate(HR)(right)onD1,D5and

D10ofheatacclimation.Dottedlinerepresentslineofequality.N=10(5M,5F).

Notes:Males(closedmarkers)Females(openmarkers)STHA(D1toD5)(o)LTHA(D1toD10)(∆).

TherewasamaineffectofdayonHRrest(F(2,16)=15.227,p≤0.001,np2=0.656).HRrestreduced

fromD1toD5(p=0.040)fromD1toD10(p=0.008)andfromD5toD10(p=0.008).Therewasno

interactioneffectbetweendayandsexonHRrest(F(2,16)=0.383,p=0.688,np2=0.046).However,

themeanreductioninHRrestfromD1toD5wasgreaterinmaleparticipants(9±10beats.min-1)

comparedwith females (6±5beats.min-1).Themeanreduction inHRrest fromD5toD10was

greaterinfemaleparticipants(6±4beats.min-1)comparedwithmales(3±2beats.min

-1).

Hsp72mRNAresponsestoheatacclimationbetweensexes

Figure7.2presentsthemeans±SDforHsp72mRNA,preandpostonD1,D5,andD10ofcontrolled

hyperthermiaHAbetweenmalesandfemales.TherewasamaineffectoftimeonHsp72mRNA

response(F(1,8)=32.998,p≤0.001,np2=0.805).Hsp72mRNAincreasedpretopostonD1(1.7±

36.0

36.5

37.0

37.5

38.0

36.0 36.5 37.0 37.5 38.0

TrerestD5andD10(°C)

Tre restD1(°C)

50

60

70

80

90

100

50 60 70 80 90 100

HRrestD5andD10(beats.m

in-1)

HRrestD1(beats.min-1)

STHAmales

STHAfemales

LTHAmales

LTHAfemales

143

0.8fold,3.9±1.8fold;p=0.003),D5(1.6±0.8fold,3.5±1.7fold;p≤0.001),andD10(2.0±0.7

fold,3.0±1.4fold;p=0.050).Therewasnointeractioneffectbetweentimeandsex(F(2,16)=1.027,

p=0.381,np2=0.114).Figure7.3presentstheindividualHsp72responsespreandpostD1,D5,and

D10relativetoD1preofcontrolledhyperthermiaHA.TheincreaseinHsp72mRNAfrompreto

postoncontrolledhyperthermiaHAD1,D5,andD10wassimilarbetweenmales(D1,1.8±1.5fold;

D5,2.0±1.0fold;D10,1.1±0.4fold)andfemales(D1,2.6±1.8fold;D5,1.8±1.4fold;D10,0.9±

1.9fold).

TherewasnomaineffectofdayonHsp72mRNAresponse(F(1,8)=0.052,p=0.826,np2=0.006).

TherewasnointeractioneffectbetweendayandsexonHsp72mRNAresponse(F(2,16)=1.027,p=

0.381,np2=0.114).Therewasnointeractioneffectbetweentime,dayandsexonHsp72mRNA

response(F(2,16)=0.479,p=0.628,np2=0.057).

Figure7.2Hsp72mRNApreandpostsessiononD1,D5,andD10ofheatacclimationinmalesand

females.Mean±SD.N=10(5M,5F).

Notes:*Denotessignificantpretopostdifferencewithinsessioninallparticipants(p≤0.05)

0

1

2

3

4

5

6

7

D1Pre D1Post D5Pre D5Post D10Pre D10Post

Hsp72m

RNA(Foldchan

ge)

Males

Females

*

*

*

144

Figure7.3Hsp72mRNApretopostsessiononD1,D5andD10relativetoD1preofheatacclimation

inmales(opencircles)andfemales(closedcircles).N=10(5M,5F).

Comparableheatacclimationsessions

Table7.3presentsthemean±SDforperformanceandphysiologicalvariablesduringD1,D5,and

D10ofcontrolledhyperthermiaHA formalesand females.Therewasnomaineffectofdayon

meanTre (F (2,16) =2.536,p =0.143,np2=0.241). Furthermore, therewasno interactioneffect

betweendayandsexonmeanTre(F(2,16)=1.880,p=0.185,np2=0.190).Therewasnomaineffect

ofdayonTrechange(F(2,16)=3.042,p=0.076,np2=0.275).Furthermore,therewasnointeraction

effectbetweendayandsexonTrechange(F(2,16)=0.234,p=0.794,np2=0.028).Therewasnomain

effect of day onmeanHR (F (2, 16) = 0.488,p = 0.623,np2= 0.057). Furthermore, therewas no

interactioneffectbetweendayandsexonmeanHR(F(2,16)=0.242,p=0.788,np2=0.029).

Therewasamaineffectofdayonmeanpower(F (2,16)=11.608,p≤0.001,np2=0.592).Mean

powerwashigheronD5(p≤0.001)andD10(p=0.015)comparedtoD1.Therewerenodifferences

betweenD5andD10(p=1.000).Therewasnointeractioneffectbetweendayandsexonmean

power(F(2,16)=0.663,p=0.529,np2=0.076).

-2

-1

0

1

2

3

4

5

6

D1Pre D1Post D5Pre D5Post D10Pre D10Post

Hsp72m

RNA(fo

ldch

ange)

Males

Females

145

Table7.3PhysiologicalandperformancemeasuresonD1,D5andD10ofcontrolledhyperthermiaheatacclimation.Mean±SD.

D1 D5 D10

Males Female Males Females Males Females

MeanTre(°C) 38.18±0.20 38.32±0.17 38.08±0.18 38.11±0.12 38.17±0.10 38.26±0.90

Trechange(°C) 1.52±0.19 1.52±0.47 1.56±0.38 1.40±0.22 1.80±0.28 1.80±0.28

MeanHR(beats.min-1) 146±14 152±9 145±8 146±10 143±12 148±9

Power(W) 87±16 62±13 121±16 81±20 127±25 87±13

Intensity(%VHO2peak) 38±5 35±8 50±6 44±7 52±11 53±14

Notes:HR,heartrate;Tre,rectaltemperature;VJO2peak,peakoxygenuptake

146

Therewasamaineffectofdayonrelativeexerciseintensity(F(2,16)=21.593,p≤0.001,np2=0.730).

RelativeexerciseintensitywashigheronD5(p≤0.001)andD10(p≤0.001)comparedtoD1.There

werenodifferencesbetweenD5andD10(p=0.221).Therewasnointeractioneffectbetweenday

andsexonrelativeexerciseintensity(F(2,16)=0.1.034,p=0.378,np2=0.114).

7.5. Discussion

ThisisthefirststudytocompareHsp72mRNAexpressioninmalesandfemalesacrossthecourse

ofcontrolledhyperthermiaHA.Incontrasttoourhypothesis,thisexperimentdemonstratesthat

theHsp72mRNAresponseissimilarbetweenmalesandfemalesonD1,D5,andD10ofacontrolled

hyperthermiaHA.ThissuggeststhatsexdifferencesinHSPfollowingacute(Gillumetal.,2013)and

chronic (Morton et al., 2009) in vivo exercise bouts are due to post transcriptional events.

ControlledhyperthermiaHAresultedintypicalphenotypicadaptations,evidencedbyreductionin

resting Tre and HR across the course of controlled hyperthermia HA. Males and females

demonstrated equal physiological responses (∆Tre,mean Tre andHR) to eachHA sessionwhere

Hsp72mRNAwasmeasured. Accordingly, equality of these endogenous stimuli, both between

groups, and throughout HA elicited equal increases in Hsp72mRNA transcription. Comparable

transcription of Hsp72mRNA betweenmales and females, suggests endogenous stimuli which

inducetheHSRarethemostimportantcriteriaforincreasingHsp72mRNA(Gibsonetal.,2015a),

withnosexdependentinhibitionoramplificationintranscription.

Inthecurrentstudy,HAproducedasignificantincreaseinHsp72mRNAprovidingfurtherevidence

thatthecontrolledhyperthermiaHAmethodpresentsasufficientendogenousstresstosurpass

theminimum requirement to elicit increased transcription of Hsp72mRNA, in bothmales and

females, at the onset and culmination of discrete and repeated bouts of exercise-heat stress.

Gibsonetal.(Gibsonetal.,2015a)reportedanincreaseinHsp72mRNApretopostonD1(1.9±

0.6fold,4.9±1.1fold),D5(2.3±0.8fold,5.3±2.5fold)andD10(2.1±0.7fold,4.3±1.3fold)

duringa10-dcontrolledhyperthermiaHAprotocol inmaleparticipants.Thedata inthecurrent

studydemonstratesfemaleshaveacomparablemagnitudeofresponsetomales.Accordingly,this

data providesmechanistic support for practitioners prescribing controlled hyperthermiaHA for

femaleathletes.SustainedincreasesinHsp72mRNAthroughouttheHA,furtherdemonstratesthe

continuedstimulationofthepathwaysresponsibleforthermotolerance,i.e.theequalityofHSR,in

bothmalesandfemales.

Previously,agreaterHSP72increasehasbeenreportedinmalescomparedtofemales(Gillumet

al.,2013;Mortonetal.,2009);however,thesestudiesmeasuredtheprotein(HSP)whereasthe

147

currentstudymeasuredthegene(HspmRNA).Interestingly,thecurrentdatacontradictsParooet

al.(Parooetal.,2002)findings,whoreportedasexspecificHSRatthelevelofproteinandmRNA;

withmaleratshavingasignificantlyhigherHSP70(200%ofcontrol)andHsp70mRNA(+900%of

control) response following 60-min of exercise at 70% V\O2max when compared with females

(HSP70=100%ofcontrol;Hsp70mRNA=450%ofcontrol).Parooetal. (Parooetal.,2002)did

however provide mechanistic evidence for an interaction between HSP70, Hsp70 mRNA and

oestrogen. Ovariectomized female animals treated with a placebo demonstrated equivalent

increases in HSP72 (+150% of control) and Hsp72mRNA (+1,200% of control) tomales, whilst

endogenousoestrogenreturnedthetypicalinhibitedfemaleHSRresponse(HSP70100%ofcontrol;

Hsp70mRNA300%ofcontrol).Methodologically,Parooetal.(Parooetal.,2002)implementedthe

northernblottingtechniquewhichislesssensitivethantheRT-QPCRtechniqueusedinthecurrent

study,potentiallyexplainingthenon-significantlyincreasedHsp72mRNA.

Elevatedoestrogenaffordscellularprotection(Shinoharaetal.,2004)andthus,thiscytoprotective

pathway may inhibit changes in HSP72 translation (Bombardier et al., 2009). It is likely, that

oestrogen most greatly mediates post Hsp72 mRNA transcriptional changes which inhibit the

translationofHSP72.ThemechanismbywhichoestrogenattenuatesHSRmaybemediatedthrough

its indirect antioxidant properties by stabilising cellular membranes and attenuating oxidative

stress;suchaneffectcouldprotectthermalsensitivecellsagainstexercise-induceddamage,and

therebyresult inabluntedHSP72response(Parooetal.,2002).Thelackofobserveddifference

betweenmalesandfemalesinthecurrentstudy,maybearesultoflowoestrogenconcentrations,

whichmaynothavebeensufficienttoexertanantioxidanteffect.

7.6. Limitations

FutureworkshouldinvolvethemeasurementofHSP72proteinalongsideHsp72mRNAacrossthe

courseofcontrolledhyperthermiaHAinmalesandfemales,tohelpunderpinthetrueeffectofsex

ontheHSR;theabsenceofwhichisalimitationofthepresentstudy.Furthermore,oestrogenis

reported to have a dose dependent inhibition of HSP72 expression at the transcription level

(Shinoharaetal.,2004).FutureworkshouldinvestigatetheHSR,andsubsequentHSP72andHsp72

mRNAresponsetodiscreteandrepeatedboutsofexercise-heatstressinhighandlowoestrogen

conditionsandinpost-menopausalwomen,whonaturallyhaveloweroestrogenconcentrations.

ThisinformationwouldhelppractitionersimplementcontrolledhyperthermiaHAstrategiesthat

ensureanoptimalstimulusforcellularadaptation.

148

7.7. Conclusion

Males and females have equal Hsp72 mRNA expression throughout 10-d of controlled

hyperthermia HA. This suggests that there are no differences in the endogenous criteria to

transcribe Hsp72 mRNA via the HSR between males and females. Differences in basal HSP72

observedelsewherearethereforelikelytoresultfrominhibitedproteintranslation,potentiallydue

totheinfluenceofoestrogen.

ThisstudyexploredthecellularadaptationsinmalesandfemalesacrossthecourseofHA.Sinceno

sex differences in the cellular adaptation were observed, Chapter 8 will take a more applied

approachtoinvestigatewhetherthephenotypicadaptationscanbeacceleratedduringSTHA.

149

8. Passiveheatexposureprecedingshort-termheatacclimationacceleratesadaptationinfemales

________________________________________________________________________________

The initial experiments have established the RHTT is repeatable (Study 1), mode specific and

sensitive to changes in thermoregulatory responses (Study2). In Study3 (Chapter6) it became

apparent that females’ responses toHA differed from those ofmales, but thiswas not due to

differencesinthermotolerance(Study4).Thisstudywillexploreapotentialmethodtoaccelerate

females’responsetoSTHAusingtheRHTTtomonitorchanges.

8.1. Abstract

ThecurrentstudyassessedwhetherSTHAprecededbyapassiveheatacceleratedheatadaptation

infemales.NinefemalesperformedtwoHAinterventionsinarandomisedorderseparatedby7-

wks. A RHTTwas performed 24-hr pre (RHTT1) and 24-hr post (RHTT2) STHA. During the HACH

intervention participants preceded the HA session by 20-min seated rest in a temperate

environmentwearing shorts anda sportsbra (20°C, 40%RH;WBGT=19°C).During theHAsauna

intervention participants preceded theHA session by 20-min seated rest in a hot environment

(50°C,30%RH;WBGT>40°C),wearingshortsandsportsbraanda100%vinylsaunasuit.Trerest(-

0.28±0.16°C),peakTre(-0.42±0.22°C),HRrest(-10±4beats.min-1),peakHR(-12±7beats.min-1),

Treattheonsetofsweating(-0.29±0.17°C)reduced(p≤0.001)fromRHTT1toRHTT2inHAsauna;

butnotHACH.TS(p=0.002)andRPE(p≤0.001)reducedfromRHTT1toRHTT2inHAsauna;butnot

HACH.PlasmavolumeexpansionwasgreaterfollowingHAsauna(9.3±7.6%)comparedtoHACH(1.3±

5.0%)(p=0.013).Sweatrateincreased(p≤0.001)andsweatsodiumchloride(NaCl)concentration

reduced (p = 0.006) in both conditions. HAsauna was effective in attenuating thermoregulatory,

cardiovascularandperceptualstrain;thiswasnotachievedfollowingHACH.ThisnovelHAstrategy

providesatime-efficientmethodtostimulateHAinfemales.

8.2. Introduction

Heat acclimation improves thermal comfort (Gonzalez and Gagge, 1976), submaximal exercise

performance, and increases maximal aerobic capacity in the heat (Lorenzo et al., 2010). The

benefits arise from enhanced sudomotor and skin blood flow responses, PV expansion,

cardiovascularstabilityandanimprovedfluidbalance(Poirieretal.,2015).Asaresult,HAisthe

consensusrecommendationstrategytoattenuatethephysiologicalstrainassociatedwithtraining

150

and competing in the heat (Racinais et al., 2015). However, there remains a paucity of data

concerningbestpractice forHA in females;consequently, femaleathletesare implementingHA

strategiesbasedondatacollectedfrommaleparticipants.Theimplicationsofthisarethatfemales

maybeadoptingsub-optimalstrategiesthatprovidelittlebenefittoalleviatingphysiologicalstrain.

Heatacclimation isoftenseparated intoSTHA (≤8-d)andLTHA (≥18-d).Approximately70%of

maximal adaptations occur following STHA evidenced by reductions in thermoregulatory and

cardiovascularstrain(Poirieretal.,2015),combinedwithanimprovedsudomotorfunction(Buono

etal.,2009b).Study3(Chapter6)presentedevidencetosuggestthatfemalesachieveonlypartial

adaptationfollowingSTHA,evidencedwithanincreaseinwholebodySWRdespitenochangesin

other classic markers of HA. Moreover, females required LTHA to establish reductions in

cardiovascularandthermoregulatorystrain.Thesefindingsarefurthersupportedbytheworkof

Sunderlandetal.(2008)whoreportednodifferenceinTreandHRfollowing4-dHAinfemales.The

absenceofevidenceconcerningeffectiveSTHAprotocols in females isproblematic forathletes,

since STHA is a preferred regime as it provides less disruption from quality training prior to

competitionthanmoretraditionalHAprotocols(Gibsonetal.,2015b).

Recently,theconceptofcombiningpassiveheatexposureswithtemperatetraininghasreceived

considerableinterest.Scoonetal.(2007)andStanleyetal.(2014)reporteda7.1%and17.8%PV

expansion,respectively,whentemperatetrainingwasfollowedbya30-minsauna(87°C)exposure.

Furthermore,Stanleyetal.(2014)reportedthatPVreachedpeakexpansion(+17.8%)followingfour

exposures;highlightingtheeffectivenessofshort-termsaunaexposure.Multiplesaunaexposures

appear to specifically augment PV expansion, which may contribute to improved myocardial

efficiency(Horowitzetal.,1986b),increasedventricularcompliance(Horowitzetal.,1986a)and

improvedmaximalcardiacoutput(Lorenzoetal.,2010);allofwhichmaytranslateintoimproved

physicalperformance.Thus,itseemsplausiblethatcombiningpassiveheatexposurewithHAmay

resultinanacceleratedadaptationinfemalesthroughtargetingPVexpansion.

Despite observing an increase in SWR in females following STHA in Study 3 (Chapter 6), SWR

remained10% lower thanmales’baselineSWR.These findingswereevidentevenwith females

potentiallyworkingatahighermetabolicheatproductionandthusevaporativerequirement,due

tothefixedintensityprotocol.Duringuncompensableheatstress,theevaporativerequirementfor

heatbalanceexceedsthemaximumpossibleheatloss(Montainetal.,1994).Protectiveclothing

exacerbates the challenge of thermoregulation because of limited water vapour permeability

across the clothing layers, decreasing the rate of heat exchange (Cheung et al., 2000). When

wearingclothing that restricts sweatevaporation,muchof thesweatmaybecomeabsorbedor

151

trapped within the clothing, altering the rate of heat transfer. Thus, it seems plausible that

stimulatingsudomotorfunctionwhilstrestrictingsweatevaporationpriortoaHAsessionthrough

theuseofprotectiveclothingmayprovidemorecompletesudomotoradaptationduetogreater

thermalburden,withoutadditionaltrainingload.

STHAoffersamoreecologicallyvalidmethodforathletescomparedwithLTHA,thusitisessential

toestablisheffectiveSTHAmethodsforafemalepopulationwhilstlimitingtheexercisestimulus.

ThisstudyaimedtoinvestigatewhetherSTHA,precededbyapassiveheatexposure,designedto

target PV expansion and sudomotor adaptation, accelerated heat adaptation in females. The

primaryhypothesiswas thatSTHAprecededbyapassiveheatexposure (HAsauna)will result ina

greaterplasmavolumeexpansioninfemalescomparedwithHAalone.Thesecondaryhypothesis

isthatHAsaunawillproduceagreatersudomotoradaptationinfemalescomparedwithHAalone.


Participants

Ninefemaleathletes(3.2)(Mean±SD;age,22±4yrs.;BM,59.33±11.55kg;stature,1.64±0.01

m;BSA,1.6±0.2m2;sumof4skinfolds,49±11mm;V\O2peak,50±4mL.kg-1.min-1)provided

writteninformedconsenttoparticipateinthecurrentstudywhichwasapprovedbytheUniversity

ofBrightonResearchEthics&GovernanceCommitteeandconductedinlinewithhealthandsafety

guidelines(3.1)andinaccordancewiththeDeclarationofHelsinki,2013.

Preliminarytesting


(3.5.). A graded exercise test using amotorised treadmill was performed to determine lactate

threshold andV\O2 peak (3.15.). In addition,V\O2 peakwasdeterminedusing a cycle ergometer

(3.14.). A regression equation was computed for the data obtained to calculate the required

intensity (65% V\O2 peak) for the experimental controlled hyperthermia HA exercise sessions

(Appendix4).Breath-by-breathexpiredmetabolicgaswasmeasuredusingonlinegasanalysis(3.9.).

Acapillarylactatesamplewascollectedandanalysedattheendofeachstageduringthelactate

thresholdtestandoncompletionoftheV\O2peaktest(3.11.).

152

Experimentaldesign

The timing of testing for female participants was conducted in line with the menstrual cycle

standardisationguidelines(3.6.).Fourparticipantsweretakingoralcontraceptive:twousedYasmin

(30µgethinylestradiol/3mgdrospirenone)andtwousedMicrogynon(30µgethinylestradiol/150

µglevonorgestrel).Hormonalstatuswasconfirmedbytakingavenousbloodsampleonthefirst

andfinaldayofeachexperimentalintervention.Alltrialswereconductedinlinewithpre-trialdiet

and exercise standardisation (3.4.). Participants provided a urine sample upon arrive to ensure

adequate hydration (3.4.). All experimental trials were performed in a hot environment (3.7.).

Exercisewasterminatedinlinewithuniversityguidelines(3.1).Therewerenoincidencesofearly

terminationofexerciseduringtheRHTTwithinthisstudy.


Participants performed twoRHTT (3.16.) on amotorised treadmill (3.15.) separated by fiveHA

sessions.ThefirstRHTTwasperformed24-hrspriortobeginningHA(RHTT1)andthesecond24-

hrsfollowing5-dHA(RHTT2).Followinga20-minstabilisationperiodinatemperateenvironment

(3.7.),measures of Tre, Tsk (3.8.), HR (3.9.), RPE, and TS (3.13.)were recorded, and participants

enteredtheenvironmentalchamber.ToweldriednudeBMwasalso recordedtocalculateSWR

(3.12.).ThroughoutthetestTre,Tsk,andHRwererecordedat5-minintervalsandRPEandTSat10-

min intervals. Mean Tsk (Equation 2.4) was subsequently calculated. Breath-by-breath expired

metabolic gas was measured using online gas analysis throughout the RHTT (3.9.). Relative

metabolicheatproduction(W.kg-1)wascalculatedinaccordancewiththeguidelinesofCramerand

Jay(2014).

ForearmsweatsampleswerecollectedduringeachoftheRHTTusingaMacroductsweatcollector

(Wescor,Logan,UT).Immediatelypriortoenteringthechambertheparticipants’skinwascleaned

withdeionizedwateranddriedimmediatelybeforesecuringtheMacroductsweatcollector.The

collectorswereheld in placeon themidpoint of the anterior forearmby aVelcro strap,which

preventedleakageandsamplecontamination.TheMacroductcollectorhasasurfaceareaof5.2

cm2.Sweatsecretedbythesweatglands is forcedfromtheductsunderhydraulicpressureand

flowsbetween theskinand theconcaveunder surfaceof theMacroductcollectorand into the

microbore tubing spiral.A small amountofbluedye ismixedwith the secretedsweatprior to

enteringthespiraltubingtoallowforvisualidentificationoftheonsetofsweating.Theforearm

sweat samples were analysed in duplicate for sweat NaCl using a sweat conductivity analyser

(SweatChek3120,Wescor,UK).

153

Heatacclimation

Inarandomised,cross-overdesign,participantscompletedtwo5-dHAinterventions(figure8.1).

Thetwointerventionswereseparatedby7-wks.toensurethedecayofadaptation(Pandolf,1998;

Poirieretal.,2015).Followinga20-minstabilisationperiod,measuresofTre(3.8.),HR(3.9.),RPE

andTS(3.13.)wererecorded,andparticipantsenteredtheenvironmentalchamber.Forthefirst

intervention(HACH)participantsenteredintoatemperateenvironment(3.7.)whilstwearingshorts

andasportsbra,forafurther20-minseatedrest.Forthesecondintervention(HAsauna)participants

enteredintoahotenvironment(50°C,30%RH;WBGT=40°C)whilstwearinga100%vinylsauna

suittoperformafurther20-minseatedrest.Thevinylsaunasuitcoveredthemajorityofthebody

with only the hands, feet, and head exposed. Following this, participants in both interventions

completedacontrolledhyperthermiaHAsession(3.14.)wearingshortsandasportsbrainahot

environment(3.7.).AllHAsessionswerecompletedonacycleergometer(3.14.).

Figure8.1Schematicoftheexperimentaldesign.

Notes:BM,bodymass;HA,heatacclimation;HR,heatrate;RH,relativehumidity;Tre,rectaltemperature.

Bloodsamplingandanalysis

Following20-minofseatedrestacapillarybloodsamplewastakenfromthefingertippriortoeach

oftheRHTTstoestimatePVexpansionusingEquation8.1(DillandCostill,1974).Capillaryblood

sampleswerecollectedinamicrocuvetteinduplicatefortheanalysisofhaemoglobinandanalysed

usingB-HaemoglobinPhotometer(HemocueLtd,Sweden).Capillarybloodsampleswerecollected

inglasscapillarytubesinduplicatefortheanalysisofhaematocritafterbeingcentrifugedat11,800

rpmfor3-min(Haemotospin1300Centrifuge,Hawksley&SonsLtd,UK).

154

Equation8.1CalculationforchangeinPlasmaVolume(∆PV)

∆PV,%=100(PVA–PVB)/PVB

Where:∆PV,changeinplasmavolume;PVA,preHAplasmavolume;PVB,postHAplasmavolume

VenousbloodsamplesweretakeninarestedstatepriortotheRHTTsandonD22ofthemenstrual

cycle.A6mLwholebloodsamplewasdrawnfromtheantecubitalfossa.Eachsamplewasdivided

into two EDTA tubes (Starstedt, Germany).Whole blood samples were centrifuged (Eppendorf

5702RCentrifuge,EppendorfUKLtd,UK)at4400rpmforaperiodof10-min.Plasmawaspipetted

(Eppendorfresearchpipettes,EppendorfUKLtd,UK)into1.5mLmicrotubes(Westernlaboratory

science,UK)andstoredat-86°C(SanyoVIPseries,SanyoElectricBiomedicalCoLtd,Japan)until

analysis. To perform the analysis, commercially available 17β-estradiol (ab108667) and

Progesterone (ab108670) immunoenzymaticassaykits (Abcamplc,UK)wereused.Quantitative

determination of plasma concentrations of 17β-estradiol and Progesterone were performed

accordingtothemanufacturer’sguidelines. Incubationof the96wellkit, includingtherequired

qualitycontrolstandardswasperformedonanorbitalplatformshaker(Titramax1000,HeidolpUK,

UK)at1.5mmorbitalvibrationandreadbyamicroplatereaderusingabsorptionat450nm(elx800,

BioTekUK,UK).Theintra-assayandinter-assayvariability,asdescribedbythemanufacturerwas

9% and 10% for 17β-estradiol and 4% and 9.3% for progesterone respectively. The lowest

detectableconcentrationof17β-estradiolandprogesterone,asdescribedbythemanufacturerwas

8.68pg.mL-1and0.05ng.mL-1,respectively.

Statisticalanalyses

AlldatawerefirstcheckedfornormalityusingtheShapiroWilktestandcorrectedforsphericity

usingtheGreenhouseGeissermethod.Atwoway(2x2)repeatedmeasuresANOVAwasperformed

to identify differences between the physiological and perceptual responses during RHTT1 and

RHTT2 between the HACH and HAsauna conditions and the physiological and HA method

characteristicsduringD1andD5ofHACHandHAsauna(3.17.4.).Whenamaineffectorinteraction

effect was found, results were followed up using a Bonferroni corrected post hoc comparison

(3.17.6.).Partialetasquared(np2)wasusedasameasureofeffectsize(3.17.5.).Apairedsamples

t-testwasperformedonthePVdata(3.17.4.).Alldatawereanalysedusingastandardstatistical

package(SPSSversion20.0,IBM,Armonk,NewYork,USA)andreportedasmean±SD.Statistical

significancewasacceptedatthelevelofp≤0.05.

155

8.4. Results

HAprotocolduringHACHandHAsauna(D1-D5)

Table8.1presentsthemean±SDdatafortheHAprotocolandphysiologicalresponsesforboth

conditions. All participants completed fiveHA sessions on consecutive days in both conditions.

DurationspentwithaTre≥38.5°Cwasonaverage52±11minacrossallsessionsinbothconditions.

DurationwithaTre≥38.5°CreducedinbothconditionsfromD1toD5(F(1,8)=8.147,p=0.021,np2

=0.505),however,nointeractioneffectbetweendayandconditionondurationwithaTre≥38.5°C

wasobserved(F(1,8)=1.488,p=0.257,np2=0.157).Nomaineffectforday(F(1,8)=5.139,p=0.053,

np2=0.391),orinteractioneffectbetweendayandconditiononexercisedurationwasobserved(F

(1,8)=2.250,p=0.172,np2=0.220).

RelativeexerciseintensityincreasedinbothconditionsfromD1toD5(F(1,8)=5.586,p=0.046,np2

=0.411),however,nointeractioneffectbetweendayandconditionwasobserved(F(1,8)=2.967,p

=0.123,np2=0.271).TotalworkincreasedinbothconditionsfromD1toD5(F (1,8)=5.593,p=

0.046,np2=0.411),however,nointeractioneffectbetweendayandconditionwasobserved(F(1,8)

=3.215,p=0.111,np2=0.287).

PhysiologicalresponsesduringHACHandHAsauna(D1–D5)

TherewasaninteractioneffectbetweendayandconditiononTrerest(F(1,8)=5.395,p=0.049,np2

=0.403).NodifferenceswereobservedbetweenconditionsonTrerestonD1(p=0.57).Trerest

reducedfromD1toD5(p=0.006)inHAsauna,butnotinHACH(p=0.268).Therewasaninteraction

effectbetweendayandconditiononmeanTre(F(1,8)=6.380,p=0.035,np2=0.444).Nodifferences

wereobservedbetweenconditionsformeanTreonD1(p=0.167).MeanTrereducedfromD1toD5

(p=0.016) in theHACHcondition,butnot inHAsauna (p=0.132).Therewasan interactioneffect

betweendayandconditiononHRrest(F(1,8)=5.924,p=0.041,np2=0.425).Nodifferenceswere

observedbetweenconditionsforHRrestonD1(p=0.251).HRrestreducedtoagreaterextent

fromD1toD5inHAsauna(p≤0.001)comparedtoHACH(p=0.009).Nomaineffectwasobservedfor

dayonmeanHR(F(1,8)=2.837,p=0.131,np2=0.262),furthermore,nointeractioneffectbetween

dayandconditionwasobserved(F(1,8)=0.059,p=0.814,np2=0.007).

SWRwashigher intheHAsaunaconditiononD1andD5comparedwithHACH(F (1,8)=21.719,p=

0.002,np2=0.731).Similarly,SWRBSAwashigherintheHAsaunaconditiononD1andD5compared

withHACH(F(1,8)=23.710,p≤0.001,np2=0.748)(table8.1).

156

Table8.1HeatacclimationprotocolandphysiologicalresponsesduringD1andD5ofHACHandHAsauna.Mean±SD.

HACHD1 HACHD5 HAsaunaD1 HAsaunaD5

Duration≥38.5°C(min) 54±13 47±8* 61±7 47±13*

ExerciseDuration(min) 63±14 69±14 59±15 71±12

MeanExerciseIntensity(%VDO2peak) 51±14 61±4* 63±5 65±10*

Totalwork(kJ) 341±108 365±134* 346±123 423±127*

Trerest(°C) 37.30±0.13 37.22±0.21 37.33±0.13 37.07±0.19*

MeanTre(°C) 38.41±0.17 38.19±0.10* 38.31±0.14 38.20±0.19

HRrest(beats.min-1) 70±5 66±7* 73±5 64±5*

MeanHR(beats.min-1) 143±9 140±11 145±7 142±8

SWR(g.h-1) 702±116 782±222 928±226+ 1009±290+

SWRBSA(g.hr-1.m2) 428±45 474±103 568117+ 624190+

Notes:HA,heatacclimation;HR,heartrate;SWR,sweatrate;SWRBSA,sweatraterelativetobodysurfacearea;Tre,rectaltemperature;VRO2peak;peakoxygenuptake.*Denotessignificant

differencewithinconditionfromD1toD5.+DenotessignificantdifferencebetweenconditionsonD1orD5.

157

Plasmaconcentrationof17βEstradiolandProgesterone

Figure8.2presentstheplasmaconcentrationsof17β-estradiolandprogesterone.Therewasamain

effectofdayonplasmaconcentrationsof17β-estradiol(F(4,32)=55.484,p≤0.001,np2=0.874).

There were no differences between the RHTT1 and RHTT2 in both conditions. However, 17β-

estradiolwashigheronD22comparedwithRHTT1andRHTT2inbothconditions.Therewasamain

effectofdayonplasmaconcentrationsofprogesterone (F (4,32)=5.983.,p=0.04,np2=0.428).

There were no differences between the RHTT1 and RHTT2 in both conditions. However,

progesteronewashigheronD22comparedwithRHTT1andRHTT2inbothconditions.Noneofthe

experimentalsessionshadtobewithdrawnorrepeatedbasedonbloodsampleresults.

Figure8.2Plasmaconcentrationsof17ß-estradiolandprogesteronepriortoRHTT1(D3)andRHTT2

(D10) in theHACH intervention (whitebars)andHAsauna intervention (blackbars)andD22of the

menstrualcycle(greybar).Mean±SD.N=9.

Notes:*denotessignificantdifferencetoD22.

Thermoregulatoryresponses

Table8.2presentsthemean±SDforthethermoregulatory,cardiovascularandperceptualvariables

duringRHTT1andRHTT2inbothconditions.Therewasan interactioneffectbetweenRHTTand

conditiononTrerest(F(1,8)=24.636,p≤0.001,np2=0.755).NodifferenceswereobservedinTre

0

5

10

15

20

25

30

35

3 10 3 10 22

Progesterone

(ng.mL⁻¹)

Dayrelativetomenses

0

50

100

150

200

250

300

3 10 3 10 22

17ßestradiol(pg.mL⁻¹)

Dayrelativetomenses

****

****

17ßestradiol Progesterone

158

restbetweenconditionsduringRHTT1(p=0.528).TrerestreducedfromRHTT1toRHTT2inHAsauna

(-0.28 ± 0.16°C;p ≤ 0.001); but notHACH (-0.07 ± 0.17°C;p = 0.277) (figure 8.3). Therewas an

interactioneffectbetweenRHTTandconditiononpeakTre(F(1,8)=18.951,p=0.002,np2=0.703).

NodifferenceswereobservedinpeakTrebetweenconditionsduringRHTT1(p=0.888).PeakTre

reducedfromRHTT1toRHTT2inHAsauna(-0.42±0.23°Cp≤0.001),butnotHACH(-0.05±0.17°C;p

=0.380).TherewasnointeractioneffectbetweenRHTTandconditiononTrechange(F(1,8)=2.274,

p=0.170,np2=0.221).

TherewasaninteractioneffectbetweenRHTTandconditionforpeakTsk(F(1,8)=7.409,p=0.026,

np2=0.481).NodifferenceswereobservedinpeakTskbetweenconditionsduringRHTT1(p=0.091).

PeakTskreducedfromRHTT1toRHTT2inHAsauna(-0.89±0.86°C;p=0.015),butnoHACH(+0.03±

0.58°C;p=0.893).

Figure8.3Rectal temperature (Tre)at5-min intervalsduringtheRHTTfor theHACH intervention

(left)andtheHAsaunaintervention(right).OpenmarkersrepresentRHTT1andtheclosedmarkers

representRHTT2.Mean±SD.N=9.

Notes:*DenotessignificantdifferencebetweenRHTT1andRHTT2

0 5 10 15 20 25 30Time(min)

RHTT1

RHTT236.636.937.237.537.838.138.438.739.039.339.6

0 5 10 15 20 25 30

T re(°C

)

Time(min)

*******

HA HAsauna

159

Table8.2Physiologicalvariablesduringbaselinetesting(RHTT1),andfollowing5-dHACHorHAsauna(RHTT2).Mean±SD.

HACHRHTT1 HACHRHTT2 HAsaunaRHTT1 HAsaunaRHTT2

Trerest(°C) 37.34±0.17 37.27±0.27 37.37±0.19 37.09±0.22*

Trepeak(°C) 39.19±0.36 39.14±0.36 39.20±0.39 38.78±0.24*

Trechange(°C) 1.85±0.36 1.87±0.32 1.83±0.40 1.69±0.24

PeakTsk(°C) 37.26±0.42 37.29±0.44 37.62±0.68 36.73±0.69*

HRrest(beats.min-1) 70±4 66±8 73±5 64±5*

HRpeak(beats.min-1) 191±8 188±9 192±10 180±8*

SWR(g.hr-1) 847±354 1253±388* 882±378 1447±287*

SWRBSA(g.hr-1.m2) 520±222 773±263* 542±242 890±193*

TS 7.5±0.5 7.5±0.5 7.5±0.5 7.0±0.5*

RPE 19±1 18±2 19±1 16±2*

Notes:HR,heartrate;RHTT,runningheattolerancetest;RPE,ratingsofperceivedexertion;SWR,sweatrate;SWRBSA,sweatraterelativetobodysurfacearea;Tre,rectaltemperature;

Tsk,skintemperature;TS,thermalsensation.*DenotessignificantdifferencetoRHTT1.

160

CardiovascularresponsestoHACHandHAsauna

TherewasaninteractioneffectbetweenRHTTandconditiononHRrest(F(1,8)=6.545,p=0.035,

np2=0.447).NodifferenceswereobservedinHRrestbetweenconditionsduringRHTT1(p=0.172).

HRrestreducedfromRHTT1toRHTT2inHAsauna(-10±4beats.min-1;p≤0.001),butnotHACH(-4±

5beats.min-1;p=0.464)(figure8.4).TherewasaninteractioneffectbetweenRHTTandcondition

onpeakHR(F(1,8)=8.983,p=0.017,np2=0.406).NodifferenceswereobservedinpeakHRbetween

conditionsduringRHTT1 (p=0.657).PeakHR reduced fromRHTT1 toRHTT2 inHAsauna (-12±7

beats.min-1;p≤0.001),butnotHACH(-3±4beats.min-1;p=0.380).

PVexpansionwasgreaterintheHAsaunacondition(9.3±7.6%)comparedwiththeHACHcondition

(1.3±3.1%)(t(8)=-3.163,p=0.013).

Figure8.4Heartrate(HR)at5-minintervalsduringtheRHTTfortheHACHintervention(right)and

theHAsauna intervention(left).OpenmarkersrepresentRHTT1andtheclosedmarkersrepresent

RHTT2.Mean±SD.N=9.

Notes:*DenotessignificantdifferencebetweenRHTT1andRHTT2.

Metabolicresponses

VYO2reducedfromRHTT1toRHTT2inbothconditions(HACH:-0.05±0.14L.min-1,HAsauna:-0.12±

0.18 L.min-1) (F (1, 8) = 10.896,p = 0.011,np2 = 0.577), however therewas no interaction effect

0 5 10 15 20 25 30Time(min)

RHTT1

RHTT260

80

100

120

140

160

180

200

220

0 5 10 15 20 25 30

HR(b

eats.m

in⁻¹)

Time(min)

*******

HA HAsauna

161

observedbetweenRHTTandcondition(F(1,8)=0.634,p=0.449,np2=0.073).Therewasnomain

effectofRHTTonRER(F(1,8)=0.139,p=0.719,np2=0.017)orinteractioneffectbetweenRHTTand

conditiononRER(F(1,8)=0.177,p=0.685,np2=0.022).Metabolicheatproductionreducedinboth

conditionsfromRHTT1toRHTT2(HACH:-0.3±0.7W.kg-1,HAsauna:-0.8±1.0W.kg-1;F(1,8)=10.896,

p = 0.011, np2 = 0.577). No interaction effect between RHTT and condition onmetabolic heat

productionwasobserved(F(1,8)=0.901,p=0.370,np2=0.101).

Sudomotorresponses

Sweatrate(F (1,8)=49.982,p≤0.001,np2=0.862)andSWRBSA(F (1,8)=52.796,p≤0.001,np2=

0.868),increasedinbothconditionsfromRHTT1toRHTT2.NointeractioneffectbetweenRHTTand

conditionwasobservedforSWR(F(1,8)=3.893,p=0.084,np2=0.327)andSWRBSA(F(1,8)=3.605,p

=0.094,np2=0.311)(figure8.5).

TherewasaninteractioneffectbetweenRHTTandconditionforsweatNaCl(F (1,8)=13.932,p=

0.006,np2=0.635).SweatNaClreducedfromRHTT1toRHTT2toagreaterextentinHAsauna(-16±

10mmol.L-1)comparedwithHACH(-5±2mmol.L-1).TherewasaninteractioneffectbetweenRHTT

andconditionforTreatonsetofsweating(F(1,8)=12.386,p=0.008,np2=0.608).Nodifferences

wereobservedinTreatonsetofsweatingbetweenconditionsduringRHTT1(HACH37.55±0.20°C,

HAsauna37.54±0.16°C;p=0.989).TheTreatonsetofsweatingreducedfromRHTT1toRHTT2in

HAsauna(-0.29±0.15°C;p≤0.001),butnotHACH(-0.08±0.17°C;p=0.137)(figure8.5).

162

Figure8.5Sweatrate(SWR)(left),sodiumchloride(NaCl)sweatconcentration(middle),andrectaltemperatureatonsetofsweating(right)duringRHTT1(xaxis)and

RHTT2(yaxis)intheHACHintervention(closedmarkers)andHAsaunaintervention(openmarkers).Dottedlinerepresentslineofequality.N=9.

36.8

37.0

37.2

37.4

37.6

37.8

38.0

36.8 37.0 37.2 37.4 37.6 37.8 38.0

Sweato

nsetRHT

T2(°C)

SweatonsetRHTT1(°C)

10

20

30

40

50

60

70

10 20 30 40 50 60 70Na

Clsw

eatcon

centratio

nRH

TT2(m

mol.L-1)

NaClsweatconcentrationRHTT1(mmol.L-1)

200

500

800

1100

1400

1700

2000

2300

200 500 800 1100 1400 1700 2000 2300

SWRRH

TT2(g.hr-1)

SWRRHTT1(g.hr-1)

HA

HAsauna

163

Perceptualresponses

TherewasaninteractioneffectbetweenRHTTandconditiononTS(F(1,8)=67.600,p≤0.001,np2=

0.894). No differences were observed in TS between conditions during RHTT1 (p = 1.000). TS

reduced from RHTT1 to RHTT2 in HAsauna (p = 0.002), but not HACH (p = 0.729). There was an

interactioneffectbetweenRHTTandconditiononRPE(F(1,8)=24.143,p=0.001,np2=0.751).No

differenceswereobservedinRPEbetweenconditionduringRHTT1(p=0.347).RPEreducedfrom

RHTT1toRHTT2inHAsauna(p≤0.001),butnotHACH(p=0.081).

8.5. Discussion

ThecurrentstudydemonstratedthatprecedingSTHAwithapassiveheatexposureacceleratesthe

adaptation in females, with HAsauna resulting in PV expansion and superior adaptation to the

sudomotorfunctioncomparedwithHAalone.Thesefindingsareinsupportoftheaimofthestudy.

Specifically,theprimaryandsecondaryhypothesiswerebothsupported. Inaddition,thesedata

confirmedthefindingsfromStudy3(Chapter6)withfemalesfailingtoachievesomeoftheclassic

markers of controlled hyperthermia STHA, including a reduced thermoregulatory and

cardiovascular strain.However, followingHAsauna reductions in thermoregulatory, cardiovascular

andperceptualstrainwereobserved.TheadaptationpathwaywaslikelymediatedinpartbyPV

expansion and an improved thermoeffector and thermosensitivity response of the sudomotor

function.TheimportanceofthesefindingsisthatHAsaunamayaccelerateHAwithoutincreasingthe

exercise stimulus.However, it remainsunknown if thereareadditionalmodifications to theHA

protocol that may offer the potential for further accelerating the adaptations within a female

population.

HACHandHAsaunaweresuccessfullymatchedfortheprescribedtrainingparametersofdurationTre

≥38.5°C,exerciseduration,relativeexerciseintensityandtotalworkcompleted(table8.1).This

providesconfidencethattheadditionaladaptationsinducedfollowingHAsaunacomparedwithHACH

werearesultofthe20-minpreHAsessionexposuretoahotwhilstwearingasaunasuitcompared

withtemperateconditions.ThedataindicatethattheHAsaunaisaneffectiveHAstrategy,evidenced

byareductioninTrerest,peakTre,HRrest,peakHR,TSandRPEcombinedwithPVexpansionand

an enhanced thermoeffector (Tre at onset of sweating) and thermosensitivity (SWR) of the

sudomotorfunction.Thesefindingsare largely inkeepingwithotherresearchemployingsimilar

STHAregimesinmales(Garrettetal.,2012;Gibsonetal.,2015b;Nealetal.,2015).

164

PlasmavolumewasunchangedintheHACHcondition.Thesefindingsareinaccordancewithother

controlledhyperthermiaSTHAinvestigationsusingmaleparticipants(Garrettetal.,2009;Nealet

al.,2015).IthasbeensuggestedthatsaunaexposureactsasanindependentstimulusforHAby

augmentingPVexpansion(Scoonetal.,2007;Stanleyetal.,2015).Inthecurrentstudy,therewas

a9.3±7.6%PVexpansionfollowingtheHAsaunacondition.ThemagnitudeofPVexpansionisvery

similartothatobservedbyothersfollowingLTHAandlong-termsaunaexposure(Burketal.,2012;

Gibsonetal.,2015c;Stanleyetal.,2015);butexceededthe6.5%expansionreportedbyLorenzo&

Minson(2010)followingLTHAand7.1%expansionreportedbyScoonetal.(2007)followinglong-

termsaunaexposure.PVexpansionresultsinanincreasedvascularfillingtosupportcardiovascular

stability, increased specific heat capacity of blood and an improved skin blood flow responses

(Sawkaetal.,2011a).Thus,thereducedthermoregulatoryandcardiovascularresponsesobserved

inthecurrentstudywerelikelyinpartmediatedbythePVexpansion.

Enhancedthermosensitivityofthesudomotorfunctionwasobserved,evidencedbya407g.hr-1and

564g.hr-1increaseinSWRduringtheRHTTfollowingtheHACHandHAsaunacondition,respectively.

These findings are in accordance with the 691 g.hr-1 increases in SWR previously observed in

females following STHA (Study 3, Chapter 6). Furthermore, in the current study, there was an

enhancedthermoeffectorresponse,evidencedbyareductionintheTreattheonsetofsweatingin

theHAsaunacondition.Thus,theobservedincreaseinSWRmayinpartbemediatedbyanincreased

cholinergicsensitivityoftheeccrinesweatglandcausinganalteredafferentneuralactivityfrom

the central thermoreceptors that altered the integration of thermal information (Charkoudian,

2010). Despite an enhanced thermosensitivity following the HACH condition there were no

alterations in the thermoeffector response, thus, these findingsmay inpartbeexplainedbyan

increaseinglandularhypertrophy(Buonoetal.,2009b;LorenzoandMinson,2010).Thereduced

thermoregulatorystrainobservedinthecurrentstudywaslikelyinpartmediatedbyanenhanced

evaporativeheatlosscombinedwithareductioninmetabolicheatproduction.

Thisstudyisthefirststudytoprovidedataonsweatmineralconcentrationsandlossesfollowing

controlledhyperthermiaSTHAinafemalepopulation.BoththeHACHandHAsaunaconditionresulted

inareductioninsweatNaCl.ThemagnitudeofreductionfortheHAsaunaconditionexceededthat

reportedpreviouslyinmalesfollowingSTHA(Nealetal.,2015).Themechanismofsweatmineral

conservationisunclear,althoughpreviousdatasuggestssweatsodiumconservationfollowingHA

involvesincreasedsodiumionreuptakewithinthere-absorptiveductofthesweatgland(Satoand

Dobson,1970; Satoet al., 1971). This is anovel findingpresentedwithin the current studyhas

practicalrelevancetofemaleathleteswhomayexperiencechronicperfusesweatingduringtraining

whichmayraisethepotentialformineraldeficiencies(Chinevereetal.,2008).

165

HAsaunaresultedinareductionpretopostinTSandRPE.Thismayhaveimplicationsforself-paced

exerciseintheheat,asaconsequenceofTSbeingamaindriverofbehaviouralthermoregulation

(FlourisandSchlader,2015).Theobservedreductions inTSandRPE followingHAsauna isanovel

finding with practical relevance for athletes preparing to compete under hot environmental

conditions.However,furtherinvestigationsarerequiredtodeterminetheeffectofSTHA,withor

withoutadditionalsaunastressonpacingandtimetrialperformanceinfemales.

8.6. Limitations

Typically, it issuggestedthatphenotypicadaptationsdecayatarateofa1-dlossofacclimation

statusforevery2-dspentwithoutheatexposure(GivoniandGoldman,1973),supportingthe7-

wks between the twoHA conditions adopted in the current study. However, evenwith 7-wks.

betweenthetwoHAconditionsitispossiblethatthemolecularmemoryfromthepriorcondition

wasdifferentbetweenparticipants(Horowitz,2014).Thecurrentstudycontrolledforthisbyusing

arandomiseddesignhowever,furtherinvestigationisrequiredtoquantifythemolecularmemory

establishedduringthetwoconditions.

ChangesinPVwereestimatedviachangesinhaematocritandhaemoglobinconcentrations.This

methodhasbeenreportedtoreflectsmall-to-moderatechangesinPVaccurately,however,itmay

underestimatelargechangesinPV(LundvallandLindgren,1998).Nevertheless,atypicalerrorasa

coefficientofvariationof4.0%and3.9%forthemeasurementofhaematocritandhaemoglobin

respectively was observed between duplicate samples providing confidence that the change

observedisameaningfulchange.

Thecurrentstudyrecruitedphysicallyactivefemaleswhoperformedbothrunningandcyclingin

theirweeklytrainingregimenandhadanaverageVdO2peakof50mL.kg-1.min-1.Thus,theseresults

areconstrainedtoaphysicallyactive,femalepopulation.HAisastrategytypicallyadoptedbyhighly

trainedathletespriortotrainingandcompetingintheheat.Furtherinvestigationsarerequiredto

establishthemagnitudeofadaptation inahighlytrainedgroupoffemaleswhomayexperience

smallermarginalgainfromHA.

8.7. Conclusion

ThisisthefirststudytoinvestigatemethodstooptimiseSTHAspecificallyforfemales.TheHAsauna

conditionestablishedreductionsinthermoregulatory,cardiovascularandperceptualstrain,andan

improved thermosensitivity and thermoeffector sudomotor function. These findings provide

evidencethatprecedingaHAsessionwithapassiveheatexposuremayofferatime-efficientmeans

166

bywhichtofurtherenhanceacclimationinafemalepopulation.Furtherinvestigationsarerequired

to establish the optimal exposure to establish the greatest magnitude of adaptation whilst

minimisingtheexercisestimulusandexposureduration.

167

9. Generaldiscussion

________________________________________________________________________________

Thischapterwillbepresentedinfourmainsections.Firstly,theprinciplefindingsforeachofthe

studiespresentedwithinthisthesiswillbediscussedwithatableofhypothesesfromeachstudy

andwhethertheywereacceptedorrejected(table9.1).Secondly,amechanisticoverviewwillbe

provided,forthemainphysiologicalissuesarisingfromtheexperimentalstudies,usingpooleddata

fromacrossallthestudiespresentedwithinthisthesis.Possibledirectionsforfutureresearchwill

thenbeconsideredpriortodiscussingthepracticalapplicationandimpactofthedatapresented

withinthisthesis.

9.1. PrincipleFindings

9.1.1. Heattolerancetest

Thefirstexperimentalstudy(Chapter4)withinthisthesisintroducedthefirstHTTwhichadoptsa

runningmodeofexerciseandassesseditsrepeatability.Thedatafromthisstudysupportedthe

primaryandsecondaryhypothesis(table9.1).PhysiologicalresponsesduringtheRHTThadstrong,

positivecorrelationsandnosignificantdifferences inphysiological responsesbetweenrepeated

trials. FollowingChapter4, the sensitivityof theRHTT to changes inheat tolerance followinga

chronic,heat-alleviatingstrategywasassessed.Thedatafromthisstudy(Study2,Chapter5)also

supported the primary and secondary hypothesis (table 9.1). Thus, the RHTT was sensitive to

changesinheattoleranceandtoindividualdifferencesinthemagnitudeofadaptation.Theresults

fromthesetwostudiesindicatethattheRHTTisasensitivetoolwhichcanbeusedtoquantifythe

changes in tolerance following chronic heat-alleviating procedures when using a repeated

measuresdesign. Inaddition, the results suggest that theRHTTmaybeanappropriate tool for

physiologists,coachesandmedicalstafftousetohelpinformdecisionsregardingathletes’safety,

and to inform the severity of the heat-alleviating procedures required prior to training and

competingintheheat.

9.1.2. Sexcomparisonstoheatacclimation

Thedatafromthethird(Chapter6)andfourth(Chapter7)studiespresentedwithinthisthesiswere

collectedtogether,butreportedastwoseparatechapterssincetheywereaddressingverydistinct

questions.Theaimofthethirdstudywastoprovidethefirstdatacomparingmales’andfemales’

phenotypicadaptationstoSTHAandLTHAusingtheRHTTtoquantifythemagnitudeofadaptation.

168

HAwaseffectiveinattenuatingphysiologicalstrainandimprovingexerciseheattolerance.STHA

waseffectiveinachievingpartialadaptationinbothmalesandfemales.However,femalesrequired

LTHA to establish reductions in cardiovascular and thermoregulatory strain. The data from this

study supports the primary, secondary and tertiary hypothesis (table 9.1) and provides novel

informationonfemales’phenotypicresponsestocontrolledhyperthermiaHA.

Theaimofthefourthstudy(Chapter7)wastoprovidethefirstdatacomparingmales’andfemales’

cellularadaptionstoSTHAandLTHAviathequantificationofHsp72mRNA.Malesandfemaleshad

similarHsp72mRNAexpressionacrossthecourseofcontrolledhyperthermiaHA.Therewereno

differences in the endogenous criteria to elicit the HSR and the adaptation towards

thermotolerancebetweenmalesandfemalesoccursindiscriminately.Thisdataisnotinsupportof

thehypothesisthatHsp72mRNAresponsewillbeattenuatedinfemalescomparedtomalesacross

thecourseofcontrolledhyperthermiaHA(table9.1).Accordingly,thisdataprovidesmechanistic

supportforpractitionersprescribingcontrolledhyperthermiaHAforfemaleathletes.

9.1.3. Acceleratedheatacclimationresponsesinfemales

Theaimofthefinalstudypresentedwithinthisthesiswastoinvestigatewhetheritispossibleto

acceleratethephenotypicadaptionstoSTHAinfemalesduringthefollicularphaseofthemenstrual

cycle,confirmedbyplasmaconcentrationsof17ßestradiolandprogesterone.Todothis,anovel

HAmethodcombiningapassivesaunalikeexposurewithcontrolledhyperthermiawasused,which

combinedcontrolledhyperthermiaHAwithapassiveexposurein50°Cwhilstwearinga100%vinyl

saunasuit.ThisinterventionresultedinanexpansionofPVandanimprovedsudomotorfunction,

resultinginareducedrestingandpeakthermoregulatoryandcardiovascularstrain,inadditionto

areductioninperceptualresponses;allofwhichwerenotobservedfollowingHAalone.Thisnovel

dataisinsupportoftheprimaryandsecondaryhypotheses(table9.1).Thesefindingsprovidenew

evidencethatfemalescanacceleratethephenotypicadaptationsduringcontrolledhyperthermia

HA.

169

Table9.1Hypothesesforeachstudychapterpresentedinthisthesis.

Hypotheses Accepted Rejected

Study1(Chapter4):Repeatabilityofarunningheattolerancetest

Physiologicalresponsesduringtherunningheattolerancetestwillhaveastrongpositivecorrelationonrepeatedtrials. X

Therewillbenodifferencesinphysiologicalresponsesduringtherunningheattolerancetestbetweenrepeatedtrials. X

Study2(Chapter5):Sensitivityofarunningheattolerancetest

TherewillbedifferencesinphysiologicalresponsesduringtherunningheattolerancetestfollowingSTHA. X

Therunningheattolerancetestwillbesensitivetoindividualdifferencesinthemagnitudeofadaptation. X

Study3(Chapter6):Acomparisonofmaleandfemaletemporalpatterningtoshort-andlong-termheatacclimation

MaleswillachievegreaterreductioninTreandHRfollowingshort-termheatacclimationcomparedwithfemales. X

TherewillbenodifferencesinthereductionsinTreandHRbetweenmalesandfemalesfollowinglong-termheatacclimation. X

Femaleswillachieveagreaterincreaseinsweatratefollowingshort-termheatacclimationandlong-termheatacclimationcompared

withmales.

X

170

Study4(Chapter7):SexcomparisonofleukocyteHsp72mRNAtranscriptionduringheatacclimation

Hsp72mRNAresponsewillbelowerinfemalescomparedtomalesacrossthecourseofcontrolledhyperthermiaheatacclimation. X

Study5(Chapter8):Passiveheatexposureprecedingshort-termheatacclimationacceleratesphenotypicadaptationinfemales

Short-termheatacclimationprecededbyapassiveheatexposurewillresultingreaterplasmavolumeexpansioninfemalescompared

withheatacclimationalone.

X

Short-termheatacclimationprecededbyapassiveheatexposurewillproduceagreatersudomotoradaptationinfemalescompared

withheatacclimationalone.

X

171

9.2. MechanisticOverview

9.2.1. HeatTolerance

ThisthesishasintroducedthefirstHTTwhichadoptsarunningmodeofexercise.Datahasbeen

presentedtodemonstratethatthemethod isrepeatable,supportingtheuseoftheRHTTwhen

usingarepeatedmeasuresdesign.Interestingly,datahasalsobeenpresentedtodemonstratethat

individuals’ thermoregulatory responses to the RHTT are highly variable with participants’

responses along a heat tolerance continuum (figure 4.1). When exercising at a fixed absolute

workload as in the RHTT, typically this results in a similar absolute heat production between

individuals of a variety of biophysical factors. However, thiswill typically result in greater heat

productionrelativetoBMinfitterindividualswithalowpercentagebodyfat,sincetheyoftenhave

asmallerbodysize.Evidencehasbeenprovidedtosupportthatwhenmatchingexerciseintensity

atafixedheatproductionrelativetototalBMthesystematicdifferencesinTcbetweengroupsof

differentbodysize(CramerandJay,2014)andfitness(Jayetal.,2011)areeliminated.Thus,the

fixedworkloadoftheRHTTmaylimitapplicabilityofthetestwhencomparingbetweenindividuals,

especiallywhentheyarenotmatchedforbiophysicalfactors.Thismaypotentiallychallengethe

RHTTbeingable todemonstrate individualdifferences inheat tolerancewith theuseofaheat

tolerancecontinuum.

To identify the predominant factors responsible for the observed differences in changes in Tre

betweenindividualsduringtheRHTTandtodeterminewhethertheprotocolhasthepotentialto

beusedtocomparebetweenindependentgroups,allfemaledatapresentedwithinthisthesis(N

=30)waspooledtoenableretrospectiveanalysis.Part1oftheanalysesinvolvedallpooleddata(N

=30).Correlationcoefficients(r)wereestablishedbetweenchanges inTreduringtheRHTTwith

BSA,sumofskinfolds,VVO2peak,relativeexerciseintensity,andheatproductionexpressedinits

absoluteform(W),relativetoBSA(W.m2),andBM(W.kg-1).

Part2of thisanalyses, involved14 femaleswhowereselected fromthepooleddata toenable

comparisonsofTrechangebetweentwoindependentgroupsoflowBM(N=7;49.5±4.5kg)and

highBM(N=7;71.7±2.2kg).ThecriteriaforselectionoftheseparticipantsforthelowBMgroup

wasaBMof≤55kgand≥65kgforthehighBMgroup.Part3oftheanalysesinvolvedaseparate

14 females, whowere selected from this pooled data to enable the comparison of Tre change

betweentwoindependentgroupsoflowVVO2peak(N=7;41±2mL.kg-1.min-1)andhighVVO2peak

(N=7;57±5mL.kg-1.min-1).ThecriteriaforselectionoftheseparticipantsforthelowVVO2peak

was≤48mL.kg-1.min-1(performancelevel1and2)andforthehighVVO2peakwas≥52mL.kg-1.min-

1 (performance level 4 and 5) (Decroix et al., 2016). Independent sample t tests were used to

172

established differences between the independent groups’ data. Participant characteristics are

displayedintable9.2andpresentedasmean,SD,minimum,andmaximumvalues.

Figure 9.1 presents the correlation coefficients for the association between changes in Tre and

biophysicalandexerciseintensityvariables.Absoluteheatproduction(r=0.603,p≤0.001;figure

9.2),heatproductionrelativetoBSA(r=0.592,p≤0.001),andheatproductionrelativetoBM(r=

0.549,p=0.002)hadmediumpositivecorrelationswithchangeinTre.BSA(r=0.349,p=0.058),

sumofskinfolds(r=0.248,p=0.186),relativeVVO2peak(r=-0.204,p=0.278),andrelativeexercise

intensity(r=0.362p=0.051)didnothaveasignificantcorrelationwithchangeinTre(figure9.1).

Trechangesweresimilarbetweenthe lowBMgroup(1.7±0.2°C)andthehighBMgroup(2.0±

0.4°C)(t(12)=1.781,p=0.100)despiteahigherheatproductioninthehighBMgroup(lowBM:515

±78W;highBM:782±176W;t(12)=3.645,p=0.003).Interestingly,whenheatproductionwas

expressedrelativetoBMthesedifferenceswereeliminated(lowBM:10.5±1.7W.kg-1;highBM:

10.9±2.6W.kg-1;t(12)=0.403,p=0.694).

TrechangesweresimilarbetweenthelowVVO2peakgroup(2.0±0.4°C)andthehighVVO2peakgroup

(1.8 ± 0.4°C) (t (12) = 0.952, p = 0.360) despite a slightly, although not significantly higher heat

productioninthelowVVO2peakgroup(lowVVO2peak:735±189W;highVVO2peak:547±143W;t

(12) = 1.617, p = 0.132). Although, when heat production was expressed relative to BM these

differenceswereeliminated(lowVVO2peak:10.7±2.4W.kg-1:highVVO2peak:10.6±2.2W.kg-1;t(12)

=0.117,p=0.909).

OncompletionoftheRHTTabsoluteheatproduction(figure9.2),heatproductionrelativetoBSA,

and heat production relative to BM were found to be the best predictors of Tre change.

Furthermore, similar changes in Tre were observed in two independent groups with large

differencesinBMandfitness.Thesefindingsareexplainedbytheexerciseintensityadministered

during the RHTT equating to similar heat production relative to BM, permitting unbiased

independentgroupcomparisonsofTc.ThesefindingsaresupportedbytheworkofCramerandJay

(2014)whoreportedsimilarchangesinTrebetweentwoindependentgroupswithlargedifferences

in BM when exercise was administered at a fixed rate of heat production relative to BM.

Furthermore,Jayetal.(2011)reportedsimilarchangesinTrewhenexercisewasadministeredat

fixedratesofheatproductionrelativetoBMbetweenindependentgroupswithlargedifferences

in fitness. These findings provide initial evidence to support the use of the RHTT between

independentgroupssinceitappearsthatdespitetheexerciseintensitybeingfixed,therelativeheat

production is similar between independent groups of different BM and fitness. These

173

interpretationsare limited toa femalepopulation, further consideration shouldbemadewhen

usingmaleparticipants.

174

Table9.2Participantcharacteristics.Mean±SD(Range).

AllData(N=30) LowBM(N=7) HighBM(N=7) LowV>O2peak(N=7) HighV>O2peak(N=7)

Stature(m) 1.66±0.08

(1.46–1.78)

1.56±0.07

(1.46-1.63)

1.71±0.05*

(1.68-1.75)

1.60±0.10

(14.8–1.70)

1.60±0.10

(1.54–1.75)

BM(kg) 60.18±8.37

(40.03-75.02)

49.51±4.52

(40.0-53.0)

71.74±2.21*

(68.0-75.0)

64.42±6.93

(51.6-72.2)

54.71±9.33

(40–70)

BSA(m2) 1.97±0.14

(1.28-1.90)

1.51±0.12

(1.28–1.56)

1.82±0.13*

(1.77–1.90)

1.74±0.11

(1.44-1.80)

1.63±0.22

(1.50–1.75)

Sumofskinfolds(mm) 56.2±23.3

(24.1–110.4)

48.1±25.0

(32.2–91.6)

73.1±24.2*

(52.0-110.2)

67.8±29.7

(24.0-110.2)

55.7±31.7

(30.0-105.6)

V>O2peak(mL.kg-1.min-1) 49±6

(34-67)

51±6

(47-57)

47±4

(42-52)

41±2

(37–44)

57±5*

(52–67)

Notes:Datapresentedfor30females.BM,bodymass;BSA,bodysurfacearea;VPO2peak,peakrateofoxygenuptake.*denotessignificantdifferencebetweenlowBMandhighBM,or

lowVPO2peakandhighVPO2peak

175

Figure9.1Correlationcoefficientsforassociationbetweenchangesinrectaltemperatureandbody

surfacearea(BSA),sumofskinfolds,relativeVCO2peak,relativeexerciseintensity,absoluteheat

production(Hprod,W),heatproductionrelativetoBSA(Hprod,W.m2),andheatproductionrelative

toBM(Hprod,W.kg-1).

Notes:*denotessignificantrelation(p≤0.05).

Figure9.2Scatterplotillustratingtherelationshipbetweenthechangeinrectaltemperature(∆Tre)

andabsoluteheatproduction(Hprod,W)duringtheRHTTinfemales.N=30.

-0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

Hprod(W.kg⁻¹)

Hprod(W.m²)

Hprod(W)

RelativeIntensity(%VO₂peak)

RelativeVO₂peak(mL.kg⁻¹.min¹)

SumofSkinFolds(mm)

BSA(m²)

CorrelationCoefficient(r)

*

*

*

y=0.0011x+1.1366r=0.60p≤ 0.05

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

0 200 400 600 800 1000 1200

∆T r

e(°C)

Hprod(W)

176

9.2.2. HeatAcclimation

Aprimaryobjectiveofthisthesiswastoexplainfemales’physiologicalandcellularresponsesto

controlled hyperthermia HA while also considering the effect of combining HA with passive

exposurestoacceleratethephysiologicalandperceptualresponsestoafixedexerciseheatstress.

Priortotheproductionofthisthesisalargebodyofresearchhadinvestigatedmales’responsesto

controlledhyperthermiaHA,however, therewasno literaturepublishedonfemales’ responses.

The limited data published on females, has used fixed (Avellini et al., 1980) or self-paced

(Sunderland et al., 2008) HA strategies which potentially constrained adaptation. This thesis

presents the first data set on females’ phenotypic and cellular adaptations to controlled

hyperthermiaHA.

Temporalpatterningofcontrolledhyperthermiaheatacclimationinfemales

Functionally,HAenhancesthecapacityofanorganismtotolerateheatstressbyaugmentingheat

dissipationandelevatingthebodytemperaturethatcanbesustainedintheheat(Horowitzand

Kodesh, 2010). Such resistance is a consequence of systemic adaptation, a chronic process

dependentuponheat-inducedreprogrammingofgeneexpression(HorowitzandKodesh,2010).

Traditionally,measuresofTre,HR,andPVwerereportedtoplateaufollowing3–8dofHA,with

SWRtaking8-14dtoreachaplateau(ArmstrongandMaresh,1991).Thisdataislimitedtomales’

responses.Therepeatedexposuretoaconstantoverloadproducesphysiologicalhabituation.Thus,

theobservedplateausinkeymarkersofHAmaybeanartefactofconstantworkratemethodsand

theinherentdecreaseinphysiologicalstrainobservedoverthedurationoftheprotocol.Toavoid

this inherent limitation and optimise adaptation, controlled hyperthermia is used. Controlled

hyperthermiaHAinvolvesrapidlyheatingdeeptissuestoatargetTc,whichisthensustainedfora

fixedduration.ControlledhyperthermiaHAensuresaprogressiveoverloadapproachwhichinduces

morecompleteadaptationwhilerevealingthemechanismsthatincreaseheattolerance(Taylor,

2014). In contrast to traditional protocols, data presented within this thesis using controlled

hyperthermiaHAdidnotresult intheclassicplateauinphysiologicalresponses.Consideringthe

responsestocontrolledhyperthermiaHAinthecurrentthesis,itislikelythatthismethodmayalter

thetemporalpatterningofadaptationwiththepotentialforsustainedadaptationastheprotocol

progresses.

177

Females’cardiovascularresponsestocontrolledhyperthermiaheatacclimation

Following controlled hyperthermia HA, reductions in resting HR have been observed in males

followingLTHA(-12beats.min-1)comparedwithSTHA(-4beats.min-1)(Garrettetal.2012;Gibson;

Gibson,Turner,etal.2015;Pattersonetal.2004a).Thiswasalsoobservedinthisthesisinfemales.

DatapresentedinStudy3(Chapter6)demonstratesanenhancedreductioninrestingHRfollowing

10-dofcontrolledhyperthermiaHA(-16beats.min-1)comparedwith5-d(-7beats.min-1)infemales.

ThesefindingsarefurthersupportedbythedatapresentedinStudy5(Chapter8)withonlysmall

reductionsinrestingHR(-4beats.min-1)observedfollowing5-dofcontrolledhyperthermiaHA.In

additiontodecreasedrestingHR,controlledhyperthermiaalsoreducedpeakHRduringtheRHTT.

Following STHA, peakHRwas reduced in both Study 3 (Chapter 6; -5 beats.min-1) and Study 5

(Chapter8;-3beats.min-1)infemaleparticipants.Anenhancedreductionwasobservedfollowing

LTHA in Study3 (Chapter 6; -10beats.min-1) and following STHA combinedwith a passiveheat

exposureinStudy5(Chapter8;-12beats.min-1).Thesefindingschallengeoriginalreportsthata

reduction in resting HR plateau following 6-d HA (Armstrong and Maresh, 1991). This further

demonstrates that the controlled hyperthermia method of HA is capable of augmenting the

physiologicaladaptationstoagreaterextentcomparedwithmoretraditionalmethods.

AreducedcardiovascularstrainfollowingHAhasbeenattributedtoanexpansionofPV.Following

controlledhyperthermiaHAanenhancedexpansionofPVisobservedinmalesfollowingLTHA(14

±1%)comparedwithSTHA(7±5%)(Garrettetal.,2014;Gibsonetal.,2015c;Pattersonetal.,2014,

2004b).InStudy5(Chapter8),PVremainedunchangedfollowingSTHA,howeverwhenSTHAwas

combined with a passive heat exposure PV increased by 9.3%, mirroring the changes in HR

responses. These findings suggest that following STHA PV has not reached full expansion,

challenging the original reports that expansion in PV plateau following 6-d HA (Armstrong and

Maresh,1991).

Females’thermoregulatoryresponsestocontrolledhyperthermiaheatacclimation

FollowingcontrolledhyperthermiaHA,anenhancedreductioninrestingTrehasbeenobservedin

males following LTHA (-0.3°C) comparedwith STHA (-0.2°C) (Garrett et al., 2014;Gibson et al.,

2015b;Neal et al., 2015; Patterson et al., 2004a).However,Gibson et al., (2015b) reported no

further reductions in restingTre following10-d (-0.09°C) comparedwith5-d (-0.10°C) controlled

hyperthermia HA. This was not observed for females in this thesis. Data presented in Study 3

(Chapter6)demonstratesnochanges in restingTre following5-d controlledhyperthermiaHA (-

0.02°C)infemales.ThesefindingsarefurthersupportedbythedatapresentedinStudy5(Chapter

178

8) with only a very small reduction in resting Tre (-0.07 °C) observed following 5-d controlled

hyperthermiaHA.However,inlinewithpreviousdataonmales,areductionwasobservedfollowing

LTHAinStudy5(Chapter8;-0.29°C)andfollowingSTHAcombinedwithpassiveheatexposurein

Study5(Chapter8;-0.28°C).AnotherclassicmarkerofHAisareducedexerciseTreowinginpart,

to a reduced resting Tre and improved heat dissipation. Following STHA, peak Tre remained

unchanged in both Study 3 (Chapter 6; -0.06°C) and Study 5 (Chapter 8; -0.05°C). However, a

reduction was observed following LTHA in Study 3 (Chapter 6; -0.47°C) and following STHA

combinedwithapassiveheatexposureinStudy5(Chapter8;-0.42°C).Thesefindingschallenge

original reports that reductions in resting Treplateau following 8-dHA (Armstrong andMaresh,

1991),withfemalesrequiringatleast10-dtoachievedreductionsinrestingTre.

Females’sudomotorresponsestocontrolledhyperthermiaheatacclimation

FollowingcontrolledhyperthermiaHAanenhancedincreaseinSWRhasbeenobservedinmales

followingLTHA(+0.46L.hr-1)comparedwithSTHA(+0.23L.hr-1)(Gibsonetal.,2015b,2015c;Neal

et al., 2015; Patterson et al., 2004a). This was also observed for females in this thesis. Data

presentedinStudy3(Chapter6)demonstratesamarginallyenhancedSWR(+0.78L.hr-1)following

10-dcontrolledhyperthermiaHAcomparedwith5-d(+0.69L.hr-1)infemales.Thesefindingsare

further supported by the data presented in Study 5 (Chapter 8) with large increases in SWR

occurringinthefirst5-dofcontrolledhyperthermiaHA(+0.41L.hr-1).Inthesefemaleparticipants,

approximately90%oftheSWRadaptationoccurredinthefirst5-dofcontrolledhyperthermiaHA.

ThesefindingschallengeoriginalreportsthatanincreaseinSWRplateausbetween8and14-dof

HA(ArmstrongandMaresh,1991)withaplateaupotentiallyoccurringmuchearlier.

The Armstrong and Maresh table for plateau days of physiological adaptation during heat

acclimatisation(ArmstrongandMaresh,1991)hasevolvedtobeatemporalpatterningmodelof

HAacquisition(table9.3).Toachieveaplateauinresponsesfollowing3-6dofHAwaspreviously

viewed favourable. However, taking into account the limitations of fixed intensity, traditional

methodsofHAandtheadvancedunderstandingofthecontrolledhyperthermiamethodofHA,the

plateaumaysimply reflect incompleteadaptation.Recent literature investigating thecontrolled

hyperthermia method of HA combined with data from this thesis on females’ responses to

controlledhyperthermiaHAaddstothetemporalpatterningmodelofHAacquisition.Controlled

hyperthermia HA ensures a progressive overload approach which induces more complete

adaptation thus, often a plateau in responses is not observed. Table 9.4 presents the days of

physiologicaladaptationduringcontrolledhyperthermiaHAinbothmalesandfemalesandusesan

adaptedformattothatofArmstrongandMaresh(1991).Thedatausedtoproducethistableisa

179

combinationofrecentcontrolledhyperthermiaHAstudies(Garrettetal.,2014,2012;Gibsonetal.,

2015b,2015c;Nealetal.,2015;Pattersonetal.,2014,2004a,2004b)anddatapresentedinthis

thesis.



AdaptationDaysofheatacclimatisation

1 2 3 4 5 6 7 8 9 10 11 12 13 14

HRdecrease ______________

PVexpansion ______________

Tredecrease ______________

RPEdecrease ______________

SweatNaCldecrease __________________

SWRincrease ___________________________

RenalNaCldecrease __________________

Notes:HR,heartrate;NaCl,SodiumChloride;PV,plasmavolume;Tre,rectaltemperature;SWR,sweatrate.

AdaptedfromArmstrongandMaresh,(1991).

Inmales,theearlyadaptationsprimarily involveanimprovedcontrolofcardiovascularfunction,

includingexpandedPVanda reducedHR. In females theearlyadaptationsprimarily involvean

increased SWR with an improved control of cardiovascular function requiring ≥10 d. Taken

together, these data would appear to suggest that when using a controlled hyperthermia HA

method the temporal patterning is different to that reported when using constant workload

protocols. The key observation is that following 10-d controlled hyperthermia HA, there is a

sustainedadaptationastheprotocolprogresses.Inaddition,thesedatawouldappeartosuggest

that females adopt a slightly different temporal patterning to males when using a controlled

hyperthermiamethodofHA.

180

Table9.4Daysofphysiologicaladaptation inmalesandfemalesduringcontrolledhyperthermia

heatacclimation.

AdaptationSex

(M/F)

Daysofheatacclimation

1 2 3 4 5 6 7 8 9 10

ReducedHRrestM _________________________________________

F ______________________

PVexpansionM _________________________________________

F ________________

ReducedTrerestM _________________________________________

F ________________

SWRIncreaseM ____________________________

F _________________________

ReducedSweatNaClM __________________________________________

F _____________________________

Notes: Solid line representsmales and thebroken line represents females.HR, heart rate;NaCl, Sodium

Chloride;PV,plasmavolume;Tre,rectaltemperature;SWR,sweatrate.

Females’acquiredthermotolerancefollowingcontrolledhyperthermiaheatacclimation

Inaddition to thephenotypicadaptations tocontrolledhyperthermiadiscussed, this thesisalso

presented the first data set on females acquired cellular thermotolerance associated with

controlled hyperthermia STHA and LTHA. The data provides confidence that when using a

controlledhyperthermiamethodofHAequalsignals for theattainmentof thermotoleranceare

presentinbothmalesandfemales.TheequalHsp72mRNAincreasesoccurringinbothmalesand

females,whowererequiredtocompletearangeofexerciseintensitiestoachievethetargetTc,

suggeststhataslongasaminimumendogenouscriterion(38.5°C)(Gibsonetal.,2014)issurpassed,

additionalexerciseorthermalstimulusisnotoffurtherbenefit.Thesefindingsareinsupportof

recent observations that controlled hyperthermia (38.5°C and 39.0°C) and fixed intensity HA

methodsallelicitsimilarHsp72mRNAresponsesacrossthecourseofLTHAassumingaminimum

181

endogenouscriteriaisachieved(Gibsonetal.,2015a).Takentogether,thesedatawouldappearto

suggestthatexerciseperseisnotasignificantindicatorofHsp72mRNAresponseandthelevelof

hyperthermiaachievedisamoreimportantdriverfortheacquiredthermotolerance.

9.2.3. Acceleratedheatacclimationinfemales

TherewasclearevidenceofHA inStudy5(Chapter8)whencontrolledhyperthermiaSTHAwas

preceded by a sauna-like exposure to 50°C whilst wearing a 100% vinyl sauna suit. This was

demonstratedbyalowerrestingTre(-0.25±0.16°C)andalowerpeakTre(-0.42±0.22°C)following

the RHTT. AdditionalHA responses included, PV expansion (+9.3 ± 7.6%), a lower set point for

sweatingonset(-0.28±0.16°C),areductioninsweatNaCl(-16±10mmol.L-1)andanincreasein

SWR(+0.57±0.20L.hr-1).Furthermore,restingHR(-10±4beats.min-1),peakHR(-12±7beats.min-1), RPE (-3 ± 1), and TS (-0.5 ± 0.5) reduced. The mechanism responsible for establishing this

acceleratedadaptationexposurerequireselucidation.

Thetypeofthethermalloadinginducedduringheatexposure,toelicitadaptation,willinfluence

thevascularadaptationthatoccurs.TheoriginalworkbyFoxetal.(1964)andlaterReganetal.

(1994)demonstratedthatwhileHAisdependentuponthedegreeofTcelevation,theelevationof

Tskisimportantforcompleteadaptation.Duringpassiveheatingcutaneousbloodflowismaximised

sincethereis lesscompetitionfortheavailablecardiacoutput.However,whenexercisinginthe

heatthereisahigherdemandfromactivemuscleforbloodsupply,thuscutaneousbloodflowis

compromised(Kenneyetal.,2014).Thisdifferenceinbloodflowdistributionmayhelpexplainthe

importanceofelevatingTskforbothitsdirectimpactonthebloodvolume(Mawetal.,2000)and

for its role within the heat adaptation process (Taylor, 2014). HA modifies both the central

thresholds and the sensitivity of cutaneous blood flow, with the former reflecting altered

hypothalamicprocessing(Bruck,1986;Horowitz,2014).Thismechanismmayinpartexplainthe

observedadaptationswhencontrolledhyperthermiaHAwasprecededbyasauna-likeexposurein

Study5(Chapter8),withalikelyincreaseinTskoccurringduringthisexposure.

A mechanism which may explain some of the observed reductions in thermoregulatory strain

followingcontrolledhyperthermiaHAcombinedwithasauna-likeexposure isPVexpansion.An

increaseinvenousreturntotheheartenhancesthesensitivityofskinvasodilationinresponseto

increasedTcduringexercise.Noseetal.(1990)reporteda35%increaseinskinbloodflowwitha

150 mL increase in PV following intravenous saline infusion when exercising in a warm

environment.However,thesefindingsarechallengedbyTakenoetal.(2001)whoexaminedthe

effectsofPVexpansiononthesensitivityoftheskinvasodilatoryresponsetoanincreaseinTcina

182

coolandwarmenvironment.Theyfoundthattheincreaseinsensitivitywaslessinacoolthanwarm

environments despite a similar PV expansion. These results suggest that improvements of the

thermoregulatoryresponseafteraerobictrainingareprimarilycausedbyneuraladaptationofthe

thermoregulatorycentreinthehypothalamustorepeatedheatexposureduringtrainingandthat

increasedPVisnotacauseofimprovement,butaresultofadaptation.

Anelevatedsensitivityofthesweatingresponsealsoaccompaniesheatadaptation,andthisitis

apparentwhenheatadaptedindividualsareeitherheatexposed(Foxetal.,1964,1963a;Gotoet

al., 2010; Lorenzo andMinson, 2010; Patterson et al., 2004a), or stimulated pharmacologically

(Collinsetal.,1966).Foxetal.(1967)suggestedthisphenomenonwasduetosweatglandtraining

since inhibition of local sweating during HA resulted in less pronounced sweat flow during

subsequentheat stimulus. Conversely,whenskin regionswere repeatedly stimulatedeitherby

localheating (Foxet al., 1964),orpharmacologically (Collinset al., 1966)without simultaneous

wholebodyheating,thenthoseregionsrespondedtoauniformthermalimpulsewithgreatersweat

flows.Lorenzo&Minson(2010)recentlyverifiedtheselocaltrainingresponseswithanincreased

sensitivityofsweatglandswhenlocallyinfusedwithacetylcholinefollowingHA.ThehigherSWR

duringthesauna-likeexposurescomparedwiththetemperateexposurepriortotheHAsessionsin

chapter 8 may in part, explain the observed adaptations. Sensitivity changes are principally a

peripheralphenomenonhowever,itseemspossiblethatanenhancedsweatingresponsefollowing

HAcouldbeattributedtoacombinationofcentralandperipheralmechanisms.

The responsible mechanism for establishing accelerated adaptation following controlled

hyperthermiaHAcombinedwithapassiveexposuretosauna-likeconditionsrequireselucidation,

but likely includes increased central thresholds and sensitivity of cutaneous blood flow, PV

expansion and an increase in sweat sensitivity. Additional data is required to establish these

mechanismsfullyandverifythesefindingstosupportthedevelopmentofoptimisedHAprotocols.

TogetherthisknowledgecanhelpsupportthedevelopmentofHAforfemalesbyensuringprotocols

targettheforcingfunctionsofHA.

9.3. Directionsforfutureresearch

ThisthesishasintroducedthefirstHTTthatadoptsarunningmodeofexercise.Therepeatability

(Study1, Chapter 4), and the sensitivity (Study2, Chapter 5) of theRHTThasbeenestablished

supporting the use of the RHTT when using a repeated measures design. Furthermore,

retrospectiveanalysiswithinthegeneraldiscussion(9.2.1.)providedevidencetosupporttheuse

oftheRHTTbetweenindependentgroupssinceitappearsthatdespitetheexerciseintensitybeing

183

fixed, the relative heat production is similar between independent groups of different BM and

fitness.FutureresearchisrequiredtoestablishtherepeatabilityandsensitivityofaRHTTthatelicits

matchedheatproductionbetweenindividuals.Thiswouldprovideconfidenceintheabilityofthe

proceduretobeusedwhenassessingthermoregulatoryresponsesbetweenindependentgroups.

Thisthesishasprovidedthefundamentalstudiestosupportfutureresearchindevelopingamore

comprehensive understanding into the mechanisms responsible for the thermoregulatory

adaptations to controlled hyperthermia HA in females. This thesis has taken a very practical

approach with the application to endurance athletes being the predominant focus. Additional

researchisrequiredtodevelopmoreoptimisedHAstrategiesforfemales,tosupporttrainingand

competitionsinhotclimates.

Inthepresentthesis,controlledhyperthermiaHAhasbeenusedtoinvestigatefemales’phenotypic

adaptations.FromthedatapresentedinStudy3(Chapter6),thismethodwasconsideredeffective

when conductedover 10-d.However, 5-dwas not sufficient to establish thermoregulatory and

cardiovascularadjustments,unlesscombinedwithanadditionalpassivethermalstimulus(Study5,

Chapter8).TheseadaptationsareinpartexplainedbyPVexpansion,thussupportingcardiovascular

adjustments, in addition to an increase in evaporative cooling. Lorenzo and Minson, (2010)

investigatedthemechanismsresponsibleforphenotypicadaptationbylocallystimulatingtheskin

withspecificconcentrationsoftheendothelium-dependentvasodilatoryacetylcholine,infusedvia

micro-dialysisandbyperformingastandardisedlocalheatingprotocol,inrestingmales.Theresults

fromthisstudyprovideconfidencethatadaptationsoccurwithintheskins’microcirculationand

sweatglandapparatusfollowingHAinmales,promotingasuperiorthermoregulatoryresponse.In

addition,nochangeswereobservedinmaximalskinbloodflowfollowingHA,demonstratingthat

theobservedchangesareattributable to improvements incutaneousvascular functionandnot

structuralchanges.Developingagreaterlevelofsophisticationwiththeproceduresandmeasures

used toassessHA responses in females,would furtherourunderstanding into themechanisms

responsiblefortheadaptationsobservedinfemales.

Within this thesis, where possible females have been tested during the follicular phase of the

menstrual cycle. Despite the convenience of studying females in the follicular phase of the

menstrualcycle,thebroaderclinicalrelevanceofthefindingsmaybelimitedsincefemalesareonly

inthispartoftheircycleforapproximately50%oftheirreproductivelives.Furthermore,intense

physical exercise has been associatedwith variousmenstrual irregularities including shortened

luteal phase, anovulation and amenorrhea (Loucks and Horvath, 1985). In addition, post-

menopausal women display substantially reduced concentration of these female hormones. It

184

remainsunclearwhetherfemales’heatadaptationiscompromisedasaresultofovarianhormonal

fluctuationsassociatedwithdifferentphasesofthemenstrualcycle,menstrualirregularitiesandin

post-menopausalwomen.Futureresearchisrequiredtoestablishthelevelofacclimationachieved

duringthedifferentphasesofthemenstrualcycleandinfemaleswithmenstrualcycleirregularities.

This informationmaysupport theoptimisationofprotocolsand the timingofadministeringHA

strategies.

Todate,thereisnoliteratureonthetimecourseofthedecayandmaintenanceofheatadaptation

in females.This informationhasahigh levelofpractical importance, toensure theadaptations

achievedremainpresentduringcompetition.Theobservationmadewithinthecurrentthesis,that

thetemporalpatterningoftheinductionisdifferentbetweenmalesandfemales,mayleadoneto

speculate that the temporalpatterningof thedecaymayalsodiffer.Traditionally, forevery2-d

withoutheatexposure,1-dofHAislost,(GivoniandGoldman,1973)withthetemporalpatterning

mirroringtheinduction(ArmstrongandMaresh,1991).Furthermore,thelengthofdecayhasalso

beensuggestedtodependuponphysicalfitnessofparticipantsandthemagnitudeofadaptation

achieved (Pandolf, 1998). Future research is required to determine whether the type of HA

determines the rate of decay since these previous observations were based on traditional HA

methods. Furthermore, research is required to explore the decay rate of adaptations achieved

followingeffectiveSTHAprotocolsinfemales.Inaddition,methodstomaintaintheadaptationsor

reducethespeedofthedecaywouldhaveahighlevelofpracticalimportanceforathletes.

ControlledhyperthermiaHAusedwithinthisthesis, isanexerciseintensivemodelwherebyTcis

rapidlyincreasedbyanexercisestimulusandmaintainedbyacombinationofexerciseandseated

rest.TherearepracticaldisadvantagesofusingexerciseHAprotocols.Theseprotocolscanbecostly

andimpracticalfornon-acclimatedindividualslivingincoolerclimates,astheircompletionrequires

accesstoanenvironmentalchamberortemporaryrelocationtoahotclimate.Furthermore,the

exercise required to ensure an adequate stimulus for adaptation may interfere with athletes

taperingpriortocompetition.Usinganinterventioninvolvingpassiveexposures,forexamplesauna

exposuresorhotwaterimmersionmayovercomeanumberofthepracticallimitationswithcurrent

exerciseHAprotocols.Theuseofasaunaexposureandhotwaterimmersionfollowingtemperate

traininghavebeenreportedtoexpandPV,resultinginareducedthermoregulatoryandperceptual

straininmales(Stanleyetal.,2015;Zurawlewetal.,2015).Researchisrequiredtoestablishthe

effectiveness of these passiveHA strategies in a female population. This body of researchmay

supportafuturelongitudinalstudywhereathletesincorporatepassiveheatexposuresduringtheir

typical temperate training. This will establish whether it is possible to maintain complete

185

acclimationthroughouttraining,avoidingtherequirementforintensiveheat-alleviatingprocedures

andtoofferprotectionduringunexpectedheatstressduringtraining.

9.4. Practicalapplicationoffindings

The RHTT presented within this thesis offers a practical method to assess thermoregulatory

responsestoagivenheatstress,specificallyinrunners,butwithapplicationtootheroccupations

suchasthemilitary.ThepracticalapplicationoftheRHTTistwofold.Firstly,datapresentedwithin

thisthesissupportstheuseoftheRHTTtoassessthermoregulatorychangesthatoccurduetoacute

andchronicheatalleviatinginterventions.Duetothelowtypicalerrorandcoefficientofvariation

associationwith the key thermoregulatory variables, researchers can have confidence that the

effect observed is a true effect and not due to error within the measure. Secondly, the data

presentedwithinthisthesissupportstheuseoftheRHTTtoobserveindividualsthermoregulatory

responsesover time,providing information tophysiologists, coachesandmedical staff tomake

decisions regarding athlete’s safety prior to training and competing in the heat. The practical

importanceoftheRHTTmaystretchasfarassupportingmedicaldecisionsaboutathletes’safety

toreturntotrainingandcompetitionfollowingexertionalheatillnessand/orassociatedsymptoms

(Johnsonetal.,2013).

Thepracticalimportanceofthedatapresentedwithinthethirdexperimentalchapteristhatthe

temporalpatterningtoHAisinpartmediatedbysexdifferences.STHAisapreferredregimefor

athletessinceitiseasiertoadoptwhensustainingqualitytrainingandtaperingperformanceinthe

weeks prior to competition. However, data presented within this thesis provides compelling

evidencethatconsiderationsneedtobemadewhenusingcontrolledhyperthermiaSTHAstrategies

withfemales.STHAmaybeeffectiveinachievingpartialadaptation,howeverfemalesrequireLTHA

to establish reductions in cardiovascular and thermoregulatory strain. Based on this evidence,

physiologists need to ensure that when supporting female athletes with heat-alleviating

interventions, they adopt an adequate number of HA sessions to ensure athletes are fully

acclimated,evidencedbyaplateau(≤5%change)inresponses.Ensuringthatanadequatethermal

stimulus is achievedwill likely support a reduction in thepossibilityofobtainingaheat-related

illnessduringtrainingorcompetitionintheheat.

The practical importance of the data presented in Study 5 (Chapter 8) is that controlled

hyperthermia STHA used in conjunction with a passive heat exposure to sauna-like conditions

provides a practical alternative to LTHA in females. This procedure resulted in reduced

cardiovascular and thermoregulatory strain similar to that observed following LTHA in Study 3

186

(Chapter 6). The use of this combined thermal stimulus provides new knowledge regarding

techniqueswhich can accelerate STHA. Themeasurement of central and peripheral sudomotor

adaptationinStudy5(Chapter8)providesusefulpractical insight intothesudomotoradaptions

that occurs following controlled hyperthermia HA. The use of these techniques over STHA in

females provides new knowledge that may permit better understanding of the possible

mechanismstoexplaintheadaptationsobserved,supportingfurtheroptimisationofHAstrategies.

Inaddition,thisdatasupportsfutureinvestigationsintothebenefitsofpassiveversusexerciseheat

exposuresinfemales.Passiveprocedureswouldbepreferabletoathletessincetherewouldbeless

interferencewithqualitytrainingandtheycouldbeincorporatedmoreeasilyintoataperingperiod

priortocompetition.

Thisdatahaspracticalimportancebeyondyoung,healthy,femaleathletes.Thefindingsfromthese

studiesmay be applied in amanner to support a range of females from both an occupational

population who are exposed to high ambient conditions combined with a high work load, in

addition to the elderly, and those with comorbid medical conditions that may inhibit their

thermoregulatoryability.Withatotalof389exertionalheatillnesscasesreportedinUKmilitary

personnelduringan88monthperiod(September2007-December2014),with236occurringinthe

UK,ofthese125innon-summermonths,heatillnesshasbecomeamajorpriorityofthemilitary

(Staceyetal.,2015).Ensuringourmilitarypersonalareadequatelypreparedfortraining inhigh

ambientconditionsisofupmostimportance.Withthecurrentconsiderationstoallowfemalesinto

groundclosecombatroles,strategieswillneedtobeimplementedtoensuretheirsafetyfroma

heat stress perspective. The information provided within this thesis could support the

implementationofbeneficialstrategiestoreducethelikelihoodoffemalesexperiencingexertional

heatillness.Furthermore,theelderlycomparedwithyoungerindividuals,storemoreheatduring

shortexposurestodryandhumidheat(Stapletonetal.,2014),consequentlythedeathratesduring

heatwavesareconsiderablyhigherintheelderly.Thedatapresentedwithinthisthesiscouldalso

beusedinamannertoinformfutureinvestigationsandheat-alleviatinginterventionsforelderly

females.

9.5. Conclusion

The RHTT introduced within this thesis, offers a simple practical tool to evaluate changes in

thermoregulatoryresponses.DatapresentedwithinthisthesissupportsathletesusingtheRHTTto

monitor changes in thermoregulation over time. To validate the use of the RHTT further, a

longitudinal study is required to assess the effectiveness of the protocol in predicting the

occurrence of exertional heat illness and supporting medical decisions regarding returning to

187

training following an exertional heat illness. Few scientific studies have examined methods to

optimise females’ adaptation during STHA, despite a number of female endurance athletes

suffering from exertional heat illness and/ or experiencing decrements in performance due to

additionalheatstrain.Thevolumeofdatapresentedwithinthisthesisobservingfemales’responses

to HA, collectively provide an original contribution to the literature. The thesis provides novel

information on the temporal patterning of HA; with females’ only achieving partial adaptation

followingSTHAusingacontrolledhyperthermiaprotocol.Inaddition,thisthesisoffersthefirstdata

set toobserve reductions in cardiovascular and thermoregulatory strain in females, following a

novelSTHAprotocol.Theinclusionofapassiveheatexposuretosauna-likeconditions,priortoa

typicalHAprotocol is an excitingnewmethodwhichhasbeen shown to accelerate adaptation

responses infemales.Further investigationsarerequiredtoexamineotherpossiblemethodsby

whichtooptimiseacclimationprotocolsforfemaleathletesanddevelopagreaterunderstanding

intothemechanismsanddifferencesthatmayoccuracrossthemenstrualcycle.Whilstexploring

these methods, it is imperative that the HA procedure remains practical and accessible to all

individuals,whether athleteswhen tapering for competition in a hot climate, ormilitarywhen

preparingfordeployment.

188

References

Altman,D.G.,Royston,P.,2006.Thecostofdichotomisingcontinuousvariables.Br.Med.J.332,1080.Armstrong,L.,Maresh,C.,1991.Theinductionanddecayofheatacclimatisationintrainedathletes.Sport.

Med.12,302–312.Armstrong, L.E., Casa, D.J.,Millard-Stafford,M.,Moran, D.S., Pyne, S.W., Roberts,W.O., 2007. American

CollegeofSportsMedicinepositionstand.Exertionalheatillnessduringtrainingandcompetition.Med.Sci.SportsExerc.39,556–572.

Armstrong,L.E.,Hubbard,R.W.,Deluca,J.P.,Christensen,E.L.,1986.Procedures,selfpacedheatacclimation,in:USArmyResearchInstituteofEnvironmentalMedicine.NatickMassachusetts.

Astrand,P.O.,Saltin,B.,1961.Oxygenuptakeduringthefirstminutesofheavymuscularexercise.J.Appl.Physiol.16,971–976.

Atkinson,G.,Nevill,A.M.,1998.Statisticalmethodsforassessingmeasurementerror(reliability)invariablesrelevanttosportsmedicine.SportMed.26,217–238.

Atkinson,G.,Reilly,T.,1996.Circadianvariationinsportsperformance.Sport.Med.21,292–312.Avellini,B.A.,Kamon,E.,Krajewski,J.T.,1980.Physiologicalresponsesofphysicallyfitmenandwomento

acclimationtohumidheat.J.Appl.Physiol.49,254–261.Bass,D.B.,Kleeman,C.R.,Quinn,M.,Henschel,A.,Hegnauer,A.H.,1955.Mechanismsofacclimatizationto

heatinman.Medicine(Baltimore).34,323–380.Bauman,J.E.,1981.Basalbodytemperature:unreliablemethodofovualtiondetection.Fertil.Steril.36,729–

733.Beaudin, A.E., Clegg,M.E.,Walsh,M.L.,White,M.D., 2009. Adaptation of exercise ventilation during an

actively-inducedhyperthermiafollowingpassiveheatacclimation.Am.J.Physiol.Regul.Integr.Comp.Physiol.297,R605–R614.

Bligh, J., 2006. A theoretical consideration of the means whereby the mammalian core temperature isdefendedatanullzone.J.Appl.Physiol.100,1332–7.

Bligh,J.,Johnson,K.G.,1973.Glossaryoftermsforthermalphysiology.J.Appl.Physiol.35,941–961.Bombardier,E.,Vigna,C., Iqbal, S., Tiidus,P.M.,Tupling,A.R.,2009.Effectsofovarian sexhormonesand

downhillrunningonfiber-type-specificHSP70expressioninratsoleus.J.Appl.Physiol.106,2009–15.Borg,G.A.V.,1962.Psychophysicalbasesofpercievedexertion.Med.Sci.SportExerc.14,337–381.Boulant,J.a,2006.NeuronalbasisofHammel’smodelforset-pointthermoregulation.J.Appl.Physiol.100,

1347–54.Brodeur,V.B.,Dennett,S.R.,Griffin,L.S.,1989.Exertionalhyperthermia,icebaths,andemergencycareatthe

FalmouthRoadRace.J.Emerg.Nurs.15,304–312.Brokenshire, C.S., Armstrong, N., Williams, C.A., 2009. The reliability of adolescent thermoregulatory

responsesduringaheatacclimationprotocol.J.Sport.Sci.Med.8,689–695.Brothers,R.M.,Bhella,P.S.,Shibata,S.,Wingo,J.E.,Levine,B.D.,Crandall,C.G.,2009a.Cardiacsystolicand

diastolicfunctionduringwholebodyheatstress.Am.J.Physiol.Hear.Circ.Physiol.296,1150–1156.Brothers,R.M.,Wingo,J.E.,Hubing,K.a,DelCoso,J.,Crandall,C.G.,2009b.Effectofwholebodyheatstress

onperipheralvasoconstrictionduringlegdependency.J.Appl.Physiol.107,1704–9.Bruck,K.,1986.Basicmechanismsinthermallong-termandshort-termadaptation.J.Therm.Biol.II,73–77.Brunt,V.E.,Miner,J.A.,Meendering,J.R.,Kaplan,P.F.,Minson,C.T.,2012.17-Bestradiolandprogesterone

independentlyaugmentcutaneousthermalhyperemiabutnotreactivehyperemia.Micocirculation18,347–355.

Budd,G.M.,2008.Wet-bulbglobetemperature(WBGT)-itshistoryanditslimitations.J.Sci.Med.Sport11,20–32.

Buono,M.,Martha, S., Heaney, J., 2009a. Peripheral sweat gland function is improvedwith humid heatacclimation.J.Therm.Biol.34,127–130.

Buono,M.,Numan,T.,Claros,R.,Brodine,S.,Kolkhorst,F.,2009b.Isactivesweatingduringheatacclimationrequired for improvements inperipheral sweatgland function?Am. J.Physiol.Regul. Integr.Comp.Physiol.297,R1082–5.

Buono,M.J.,Heaney,J.H.,Canine,K.M.,1998.Acclimationtohumidheatlowersrestingcoretemperature.Am.J.Physiol.274,R1295–R1299.

Burk,A., Timpmann,S.,Kreegipuu,K., Tamm,M.,Unt,E.,Oöpik,V.,2012.Effectsofheatacclimationonendurancecapacityandprolactin response toexercise in theheat.Eur. J.Appl.Physiol.112,4091–4101.

189

Byrne,C.,Lim,C.L.,2007.Theingestibletelemetricbodycoretemperaturesensor:areviewofvalidityandexerciseapplications.Br.J.SportsMed.41,126–133.

Cabanac,M.,2006.Adjustablesetpoint:tohonorHaroldT.Hammel.J.Appl.Physiol.100,1338–46.Calzone,W.L.,Silva,C.,Keefe,D.L.,Stachenfeld,N.S.,2001.Progesteronedoesnotalterosmoticregulation

ofAVP.Am.J.Physiol.Regul.Integr.Comp.Physiol.281,2011–2020.Carpenter,A.J.,Nunneley,S.A.,1988.Endogenoushormonessubtlyalterwomens’responsetoheatstress.J.

Appl.Physiol.65,2313–2317.Casa,D.J.,Guskiewicz,K.M.,Anderson,S.A.,Heck,J.F.,Jimenez,C.C.,McDermott,B.P.,Miller,M.G.,Stearns,

R.L., Swartz, E.E., Walsh, K.M., 2012. National athletic trainers’ association position statement:Preventingsuddendeathinsports.J.Athl.Train.47,96–118.

Castle, P.,Mackenzie, R.W.,Maxwell, N.,Webborn, A.D.J.,Watt, P.W., 2011. Heat acclimation improvesintermittentsprintingintheheatbutadditionalpre-coolingoffersnofurtherergogeniceffect.J.SportsSci.29,1125–34.

Castle,P.C.,Macdonald,A.L.,Philp,A.,Webborn,A.,Watt,P.W.,Maxwell,N.S.,2006.Precoolinglegmuscleimprovesintermittentsprintexerciseperformanceinhot,humidconditions.J.Appl.Physiol.100,1377–1384.

Chapman, A.B., Zamudio, S., Woodmansee, W., Merouani, A., Osorio, F., Johnson, A.N.N., Moore, L.G.,Dahms,T.,Coffin,C.,Abraham,W.T.,Schrier,R.W.,1997.Systemicandrenalhemodynamicchangesinthelutealphaseofthemenstrualcyclemimicearlypregnancy.Am.J.Physiol.273,777–782.

Charkoudian, N., 2010. Mechanisms and modifiers of reflex induced cutaneous vasodilation andvasoconstrictioninhumans.J.Appl.Physiol.109,1221–8.

Charkoudian,N.,2001.Influencesoffemalereproductivehormonesonsympatheticcontrolofthecirculationinhumans.Clin.Auton.Res.11,295–301.

Charkoudian,N.,Halliwill, J.R.,Morgan,B.J.,Eisenach, J.H., Joyner,M.J.,2003. Influencesofhydrationonpost-exercisecardiovascularcontrolinhumans.J.Physiol.552,635–44.

Charkoudian,N.,Johnson,J.M.,2000.Femalereproductivehormonesandthermoregulatorycontrolofskinbloodflow.Exerc.SportSci.Rev.28,108–112.

Cheung, S.S., McLellan, T.M., Tenaglia, S., 2000. The thermophysiology of uncompensable heat stress:Physiologicalmanipulationsandindividualcharacteristics.Sport.Med.29,329–59.

Cheuvront,S.N.,Ely,B.R.,Kenefick,R.W.,Sawka,M.N.,2010.Biologicalvariationanddiagnosticaccuracyofdehydrationassessmentmarkers.Am.J.Clin.Nutr.92,565–573.

Cheuvront, S.N., Kolka,M. a, Cadarette, B.S., Montain, S.J., Sawka,M.N., 2003. Efficacy of intermittent,regionalmicroclimatecooling.J.Appl.Physiol.94,1841–8.

Chinevere,T.D.,Kenefick,R.W.,Cheuvront,S.N.,Lukaski,H.C.,Sawka,M.N.,2008.Effectofheatacclimationonsweatminerals.Med.Sci.SportExerc.40,886–91.

Chomczynski, P., Sacchi, N., 1987. Single-stepmethod of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction.Anal.Biochem.162,156–159.

Chung,S.C.,Goldfarb,A.H.,Jamurtas,A.Z.,Hegde,S.S.,Lee,J.,1999.Effectofexerciseduringthefollicularandlutealphasesonindicesofoxidativestressinhealthywomen.Med.Sci.SportExerc.31,409–413.

Cohen,J.,1988.Astatisticalpoweranalysisforthebehaviouralsciences.RoutledgeAcademic,NewYork.Collins,K.J.,Crockford,G.W.,Weiner,J.S.,1966.Thelocaltrainingeffectofsecretoryactivityontheresponse

ofeccrinesweatglands.J.Physiol.184,203–214.Cooper,K.E.,Cranston,W.I.,Snell,E.S.,1964.Temperatureintheexternalauditorymeatusasanindexof

centraltemperaturechanges.J.Appl.Physiol.19,1032–1034.Cotter,J.D.,Patterson,M.J.,Taylor,N.A.S.,1997.Sweatdistributionbeforeandafterrepeatedheatexposure.

Eur.J.Appl.Physiol.76,181–6.Coyne,M.D.,Kesick,C.M.,Doherty,T.J.,Kolka,M.a,Stephenson,L.a,2000.Circadianrhythmchangesincore

temperatureoverthemenstrualcycle:methodfornoninvasivemonitoring.Am.J.Physiol.Regul.Integr.Comp.Physiol.279,R1316–20.

Cramer,M.N.,Bain,A.R., Jay,O.,2012.Localsweatingontheforehead,butnotforearm, is influencedbyaerobicfitnessindependentlyofheatbalancerequirementsduringexercise.Exp.Physiol.97,572–82.

Cramer, M.N., Jay, O., 2014. Selecting the correct exercise intensity for unbiased comparisons ofthermoregulatoryresponsesbetweengroupsofdifferentmassandsurfacearea.J.Appl.Physiol.116,1123–32.

Crandall,C.G.,Shibasaki,M.,Wilson,T.E.,2010. Insufficientcutaneousvasoconstriction leadinguptoandduringsyncopalsymptomsintheheatstressedhuman.Am.J.Physiol.HeartCirc.Physiol.299,H1168–73.

DeSouze,M.J.,Miller,B.E.,Loucks,A.B.,Luciano,A.A.,Pescatello,L.S.,Campbell,C.G.,Lasley,B.L.,1998.High

190

frequencyoflutealphasedeficiencyandanovulationinrecreationalwomenrunners:Bluntedelevationinfollicle-stimulatinghormoneobservedduringlutealfolliculartransition.J.Clin.Endocrinol.Metab.83,4220–4232.

Decroix,L.,DePauw,K.,Foster,C.,Meeusen,R.,2016.GuidelinestoClassifyFemaleSubjectGroupsinSport-ScienceResearch.Int.J.SportsPhysiol.Perform.11,204–213.

DelCoso,J.,Estevez,E.,Mora-Rodriguez,R.,2009.Caffeineduringexerciseintheheat:Thermoregulationandfluid-electrolytebalance.Med.Sci.Sport.Exerc.41,164–173.

DeMartini,J.K.,Casa,D.J.,Belval,L.N.,Crago,A.,Davis,R.J.,Jardine,J.J.,Stearns,R.L.,2014.Environmentalconditionsandtheoccurrenceofexertionalheatillnessesandexertionalheatstrokeatthefalmouthroadrace.J.Athl.Train.49,478–485.

Denis,P.,Cazor,J.L.,Feret,J.,Weisang,E.,Lefrancois,1976.2,3Diphosphoglycerateredcellconcentrationchangesduringthemenstrualcycleinwomen.Biomedicine25,144.

Dill,D.B.,Costill,D.L.,1974.Calculationofpercentagechangesinvolumesofblood,plasma,andredcellsindehydration.J.Appl.Physiol.37,247–8.

Dill,D.B.,Jones,B.F.,Edwards,H.T.,Oberg,S.A.,1933.Salteconomyinextremedryheat.J.Biol.Chem.100,755–767.

Drew,B.G.,Ribas,V.,Le,J.A.,Henstridge,D.C.,Phun,J.,Zhou,Z.,Soleymani,T.,Daraei,P.,Sitz,D.,Vergnes,L.,Wanagat,J.,Reue,K.,Febbraio,M.A.,Hevener,A.L.,2014.HSP72isamitochondrialstresssensorcriticalforParkinaction,oxidativemetabolism,andinsulinsensitivityinskeletalmuscle.Diabetes63,1488–1505.doi:10.2337/db13-0665

Druyan,A.,Makranz,C.,Moran,D.,2012.Heattolerance inwomen-reconsideringthecriteria.Aviat.Sp.Environ.Med.83,53–60.

DuBois,D.,DuBois,E.F.,1916.Aformulatoestimatetheapproximatesurfaceareaifheightandweightbeknown.Ann.Intern.Med.17,863–871.

Durnin,J.V.G.,Womersley,J.,1974.Bodyfatassessedfortotalbodydensityanditsestimationfromskinfoldthickness:Measurementson481menandwomenaged16-72years.Br.J.Nutr.32,77.

Edwards,R.J.,Belyavin,A.J.,Harrison,M.H.,1978.Coretemperaturemeasurementinman.Aviat.Sp.Environ.Med.49,1289–1294.

Eichna,L.W.,Parl,C.R.,Nelson,N.,Horvath,S.M.,Palmes,E.D.,1950.Threeaspectsofthermalbalancewerestudied:Am.J.Physiol.163,585–597.

ElHelou,N.,Tafflet,M.,Berthelot,G.,Tolaini, J.,Marc,A.,Guillaume,M.,Hausswirth,C.,Toussaint, J.-F.,2012.Impactofenvironmentalparametersonmarathonrunningperformance.PLoSOne7,e37407.

Ely,B.R.,Ely,M.R.,Cheuvront,S.N.,Kenefick,R.W.,Degroot,D.W.,Montain,S.J.,2009.Evidenceagainsta40degreesCcoretemperaturethresholdforfatigueinhumans.J.Appl.Physiol.107,1519–25.

Epstein,Y.,1990.Heatintolerance:predisposingfactororresidualinjury?Med.Sci.SportExerc.22,29–35.Epstein,Y.,Moran,D.S.,Shapiro,Y.,Sohar,E.,Shemer,J.,1999.Exertionalheatstroke:acaseseries.Med.

Sci.SportExerc.31,224–228.Epstein, Y., Shapiro, Y., Shai, B., 1983. Role of surface area-to-mass ratio and work efficiency in heat

intolerance.J.Appl.Physiol.54,831–836.Farrokhyar,F.,Reddy,D.,Poolman,R.W.,Bhandari,M.,2013.Whyperformapriorisamplesizecalculation?

Can.J.Surg.56,207–213.doi:10.1503/cjs.018012Febbraio,M.a,Koukoulas,I.,2000.HSP72geneexpressionprogressivelyincreasesinhumanskeletalmuscle

duringprolonged,exhaustiveexercise.J.Appl.Physiol.89,1055–1060.Fiala,D.,Havenith,G.,Bröde,P.,Kampmann,B.,Jendritzky,G.,2012.UTCI-Fialamulti-nodemodelofhuman

heattransferandtemperatureregulation.Int.J.Biometeorol.56,429–441.Field,A.,2013.DiscoveringstatisticsusingIBMSPSSstatistics,4thed.Sage,London.Flouris,a.D.,Schlader,Z.J.,2015.Humanbehavioralthermoregulationduringexerciseintheheat.Scand.J.

Med.Sci.Sports25,52–64.Flouris,A.D.,2011.Functionalarchitectureofbehaviouralthermoregulation.Eur.J.Appl.Physiol.111,1–8.Flouris,A.D.,Poirier,M.P.,Bravi,A.,Wright-Beatty,H.E.,Herry,C.,Seely,A.J.,Kenny,G.P.,2014.Changesin

heart rate variability during the induction anddecayof heat acclimation. Eur. J. Appl. Physiol. 114,2119–28.

Forman,R.G.,Chapman,M.C.,Steptoe,P.C.,1987.Theeffectofendogenousprogesteroneonbasalbodytemepratureinstimulatedovariancycles.Hum.Reprod.2,631–634.

Fox,R.H.,Goldsmith,R.,Hampton,I.F.,Hunt,T.J.,1967.Heatacclimatizationbycontrolledhyperthermiainhot-dryandhot-wetclimates.J.Appl.Physiol.22,39–46.

Fox,R.H.,Goldsmith,R.,Hampton,I.F.G.,Lewis,H.E.,1964.Thenatureoftheincreaseinsweatingcapacityproducedbyheatacclimatization.J.Physiol.171,368–376.

191

Fox,R.H.,Goldsmith,R.,Kidd,D.J.,Lewis,H.E.,1963a.Acclimatizationtoheatinmanbycontrolledelevationofbodytemperature.J.Physiol.166,530–547.

Fox,R.H.,Goldsmith,R.,Kidd,D.J.,Lewis,H.E.,1963b.Bloodflowandotherthermoregulatorychangeswithacclimatizationtoheat.J.Physiol.548–562.

Frank,A.,Belokopytov,M.,Moran,D.,Shapiro,Y.,Epstein,Y.,2001.Changesinheartratevariabilityfollowingacclimationtoheat.J.BasicClin.Physiol.Pharmacol.12,19–32.

Fritzsche, R.G., Coyle, E.F., 2000. Cutaneous blood flow during exercise is higher in endurance-trainedhumans.J.Appl.Physiol.88,738–744.

Fritzsche,R.G.,Switzer,T.W.,Hodgkinson,B.J.,Coyle,E.F.,1999.Strokevolumedeclineduringprolongedexerciseisinfluencedbytheincreaseinheartrate.J.Appl.Physiol.86,799–805.

Fujii,N.,Honda,Y.,Ogawa,T.,Tsuji,B.,Kondo,N.,Koga,S.,Nishiyasu,T.,2012.Short-termexercise-heatacclimationenhancesskinvasodilationbutnothyperthermichyperpneainhumansexercisinginahotenvironment.Eur.J.Appl.Physiol.112,295–307.

Gagge,A.P.,Stolwijk, J.A.J., Saltin,H.,1969.Comfortand thermal sensationsandassociatedphysiologicalresponsesduringexerciseatvariousambienttemperatures.Environ.Res.2,209–229.

Gagnon,D.,Dorman,L.E.,Jay,O.,Hardcastle,S.,Kenny,G.P.,2009.Coretemperaturedifferencesbetweenmalesandfemalesduringintermittentexercise:physicalconsiderations.Eur.J.Appl.Physiol.105,453–61.

Gagnon,D.,Jay,O.,Kenny,G.P.,2013.Theevaporativerequirementforheatbalancedetermineswhole-bodysweatrateduringexerciseunderconditionspermittingfullevaporation.J.Physiol.591,2925–35.

Gagnon, D., Jay, O., Lemire, B., Kenny, G.P., 2008. Sex-related differences in evaporative heat loss: theimportanceofmetabolicheatproduction.Eur.J.Appl.Physiol.104,821–9.

Gagnon, D., Kenny, G.P., 2012. Sex differences in thermoeffector responses during exercise at fixedrequirementsforheatloss.J.Appl.Physiol.113,746–57.

Gagnon,D.,Kenny,G.P.,2011.Sexmodulateswhole-bodysudomotorthermosensitivityduringexercise.J.Physiol.589,6205–17.

Galloway,S.D.R.,Maughan,R.J.,1997.Effectsofambienttemperatureonthecapacitytoperformprolongedcycleexerciseinman.Med.Sci.SportExerc.29,1240–1249.

Garrett,aT.,Goosens,N.G.,Rehrer,N.J.,Patterson,M.J.,Harrison,J.,Sammut,I.,Cotter,J.D.,2014.Short-termheatacclimationiseffectiveandmaybeenhancedratherthanimpairedbydehydration.Am.J.Hum.Biol.26,311–20.

Garrett,A.T., Creasy,R., Rehrer,N.J., Patterson,M.J., Cotter, J.D., 2012. Effectivenessof short-termheatacclimationforhighlytrainedathletes.Eur.J.Appl.Physiol.112,1827–37.

Garrett,A.T.,Goosens,N.G.,Rehrer,N.J.,Rehrer,N.G.,Patterson,M.J.,Cotter,J.D.,2009.Inductionanddecayofshort-termheatacclimation.Eur.J.Appl.Physiol.107,659–670.

Garrett, A.T., Rehrer, N.J., Patterson,M.J., 2011. Induction and decay of short-term heat acclimation inmoderatelyandhighlytrainedathletes.Sport.Med.41,757–771.

Geddes,L.A.,Baker,L.E.,1967.Thespecificresistanceofbiologicalmaterial-acompendiumofdataforthebiomedicalengineerandphysiologist.Med.Biol.Eng.5,271–293.

Gibson, O.R., Dennis, A., Parfitt, T., Taylor, L., Watt, P.W., Maxwell, N.S., 2014. Extracellular Hsp72concentration relates to aminimum endogenous criteria during acute exercise-heat exposure. CellStressChaperones19,389–400.

Gibson,O.R.,Mee,J.A.,Taylor,L.,Tuttle,J.A.,Watt,P.W.,Maxwell,N.S.,2015a.Isothermicandfixed-intensityheatacclimationmethodselicitequalincreasesinHsp72mRNA.Scand.J.Med.Sci.Sports25,259–268.

Gibson,O.R.,Mee,J.A.,Tuttle,J.A.,Taylor,L.,Watt,P.W.,Maxwell,N.S.,2015b.Isothermicandfixedintensityheatacclimationmethodsinducesimilarheatadaptationfollowingshortandlong-termtimescales.J.Therm.Biol.49-50,55–65.

Gibson,O.R.,Turner,G.,Tuttle,J.A.,Taylor,L.,Watt,P.W.,Maxwell,N.S.,2015c.HeatAcclimationattenuatesphysiologicalstrainandtheHsp72,butnotHsp90αmRNAresponsetoacutenormobarichypoxia.J.Appl.Physiol.119,889–899.

Gillum,T.,Kuennen,M.,Gourley,C.,Dokladny,K.,Schneider,S.,Moseley,P.,2013.Sexdifferencesinheatshockprotein72expressioninperipheralbloodmononuclearcellstoacuteexerciseintheheat.Int.J.Endocrinol.Metab.11,8739.

Gisolfi,C.V.,Wenger,C.B.,1984.Temperature regulationduringexercise:oldconcepts,new ideas.Exerc.SportSci.Rev.12,339–372.

Givoni, B., Goldman, R.F., 1973. Predicting effects of heat acclimatization on heart rate and rectaltemperature.J.Appl.Physiol.35,875–879.

Gledhill,N.,Jamnik,R.,1994.Enduranceathletes’stokevolumedoesnotplateau:Majoradvantageisdiastolic

192

function.Med.Sci.SportExerc.26,1116–1121.Gonzalez-Alonso,J.,2003.Reductionsinsystemicandskeletalmusclebloodflowandoxygendeliverylimit

maximalaerobiccapacityinhumans.Circulation107,824–830.González-Alonso,J.,Crandall,C.G.,Johnson,J.M.,2008.Thecardiovascularchallengeofexercisingintheheat.

J.Physiol.586,45–53.Gonzalez,R.R.,Gagge,A.P.,1976.Warmdiscomfortandassociatedthermoregulatorychangesduringdryand

humidheatacclimatization.Isr.J.Med.Sci.12,804–807.Goto, M., Okazaki, K., Kamijo, Y., Ikegawa, S., Masuki, S., Miyagawa, K., Nose, H., 2010. Protein and

carbohydratesupplementationduring5-dayaerobictrainingenhancedplasmavolumeexpansionandthermoregulatoryadaptationinyoungmen.J.Appl.Physiol.109,1247–55.

Grucza,R.,Pekkarinen,H.,Titov,E.K.,Kononoff,A.,Hanninen,O.,1993.Influenceofthemenstrualcycleandoralcontraceptivesonthermoregulatoryresponsestoexerciseinyoungwomen.Eur.J.Appl.Physiol.67,279–285.

Guy, J.H., Deakin, G.B., Edwards, A.M., Miller, C.M., Pyne, D.B., 2015. Adaptation to hot environmentalconditions: an exploration of the performance basis, procedures and future directions to optimiseopportunitiesforeliteathletes.Sport.Med.45,303–11.

Hammel, H.T., Jackson, D.C., Stolwijk, J.A.J., Hardy, D., Stromme, S.B., 1963. Temperature regulation byhypothalamicproportionalcontrolwithanadjustablesetpoint.J.Appl.Physiol.18,1146–1154.

Harper-Smith, A.D., Crabtree, D.R., Bilzon, J.L.J.,Walsh, N.P., 2010. The validity of wireless iButtons andthermistorsforhumanskintemperaturemeasurement.Physiol.Meas.31,95–114.

Harrison,M.H.,1985.Effectsonthermalstressandexerciseonbloodvolumeinhumans.Physiol.Rev.65,149–209.

Havenith, G., 2001. Human surface tomass ratio and body core temperature in exercise heat stress—aconceptrevisited.J.Therm.Biol.26,387–393.

Havenith, G.,Middendorp, H. van, 1990. The relative influence of physical fitness, acclimatization state,anthropometricmeasuresandgenderonindividualreactionstoheatstress.Eur.J.Appl.Physiol.Occup.Physiol.61,419–427.

Hawes,R.,McMorran,J.,Vallis,C.,2010.Exertionalheatillnessinhalfmarathonrunners:experiencesoftheGreatNorthRun.Emerg.Med.J.27,866–867.

Hayden, G.,Milne, H.C., Patterson,M.J., Nimmo,M. a, 2004. The reproducibility of closed-pouch sweatcollectionandthermoregulatoryresponsestoexercise-heatstress.Eur.J.Appl.Physiol.91,748–51.

Hayward,J.S.,Eckerson,J.D.,Kemna,D.,1984.Thermalandcardiovascularchangesduringthreemethodsofresuscitationfrommildhypothermia.Resuscitation11,21–33.

Hessemer,V.,Bruck,K.,1985.Influenceofmenstrualcycleonthermoregulatory,metabolic,andheartrateresponsestoexerciseatnight.J.Appl.Physiol.59,1911.

Hopkins,W.G.,2000a.Reliabilityfromconsecutivepairsoftrials.Sport.Sci.4.Hopkins,W.G.,2000b.Measuresofreliabilityinsportsmedicineandscience.Sport.Med.30,1–15.Horowitz,M.,2014.Heatacclimation,epigenetics,andcytoprotectionmemory.Compr.Physiol.4,199–230.Horowitz,M.,2003.Matchingthehearttoheat-inducedcirculatoryload:heat-acclimatoryresponses.News

Physiol.Sci.18,215–21.Horowitz,M.,Kodesh,E.,2010.Molecularsignalsthatshapetheintegrativeresponsesoftheheat-acclimated

phenotype.Med.Sci.Sport.Exerc.42,2164–2172.Horowitz,M.,Peyser,Y.M.,Muhlrad,A.,1986a.Alterationsincardiacmyosinisoenxymesdistributionasan

adaptationtochronicenvironmentalheatstressintherat.J.Mol.Cell.Cardiol.18,511–515.Horowitz,M.,Robinson,S.D.M.,2007.Heatshockproteinsandtheheatshockresponseduringhyperthermia

anditsmodulationbyalteredphysiologicalconditions.Prog.BrainRes.162,433–446.Horowitz,M.,Shimoni,Y.,Parnes,S.,Gotsman,M.S.,Hasin,Y.,1986b.Heatacclimation:cardiacperformance

ofisolatedratheart.J.Appl.Physiol.60,9–13.Horvath,S.M.,Drinkwater,B.L.,1982.Thermoregulationandthemenstrualcycle.Aviat.Sp.Environ.Med.

53,790–794.Houmard, J.A., Costill, D.L., Davis, J.A.,Mitchell, J.B., Pascoe, D.D., Robergs, R.A., 1990. The Influence of

exerciseintensityonheataccliamtionintrainedsubjects.Med.Sci.SportExerc.22,615–620.Howe,A.S.,Boden,B.P.,2007.Heat-relatedillnessinathletes.Am.J.SportsMed.35,1384–95.Ilsley,A.H.,Rutten,A.J.,Runciman,W.B.,1983.Anevaluationofbodytemperaturemeasurement.Anaestheic

IntensiveCare11,31–39.Inoue,Y.,Havenith,G.,Kenney,W.L.,Loomis,J.L.,Buskirk,E.R.,1999.Exercise-andmethylcholine-induced

sweatingresponses inolderandyoungermen:effectofheatacclimationandaerobic fitness. Int. J.Biometeorol.42,210–6.

193

Inoue,Y.,Tanaka,Y.,Omori,K.,Kuwahara,T.,Ogura,Y.,Ueda,H.,2005.Sex-andmenstrualcycle-relateddifferencesinsweatingandcutaneousbloodflowinresponsetopassiveheatexposure.Eur.J.Appl.Physiol.94,323–32.

James,C.A.,Richardson,A.J.,Watt,P.W.,Maxwell,N.S.,2014.Reliabilityandvalidityof skin temperaturemeasurementbytelemetrythermistorsandathermalcameraduringexercise intheheat.J.Therm.Biol.45,141–149.

JansedeJonge,X.A.K.,2003.Effectsofthemenstrualcycleonexerciseperformance.Sport.Med.33,833–51.

Jay,O.,Bain,A.R.,Deren, T.M., Sacheli,M.,Cramer,M.N.,Hi,V., 2011. Largedifferences inpeakoxygenuptakedonotindependentlyalterchangesincoretemperatureandsweatingduringexercise.Am.J.Physiol.Regul.Integr.Comp.Physiol.301,832–841.

Jay,O.,Kenny,G.,2007.Thedeterminationofchangesinbodyheatcontentduringexerciseusingcalorimetryandthermometry.J.Human-EnvironmentalSyst.10,19–29.

Johnson,E.C.,Kolkhorst,F.W.,Richburg,A.,Schmitz,A.,Martinez,J.,Armstrong,L.E.,2013.Specificexerciseheatstressprotocol foratriathlete’sreturnfromexertionalheatstroke.Curr.SportsMed.Rep.12,106–9.

Johnson,J.M.,Kellogg,D.L.,2010.Localthermalcontrolofthehumancutaneouscirculation.J.Appl.Physiol.109,1229–38.

Jones,A.,Doust,J.,1998.Thevalidityofthelactateminimumtestfordeterminationofthemaximallactatesteadystate.Med.Sci.SportExerc.30,1304–1313.

Jones,A.M.,2007.Middleand longdistancerunning, in:Winter,E.M., Jones,A.M.,RichardDavison,R.C.,Bromley,P.D.,Mercer,T.H.(Eds.),SportandExercisePhysiologyTestingGuidelines.RoutledgeTaylorandFrancisGroup,LondonandNewYork,pp.147–154.

Jones,A.M.,Doust,J.H.,1996.A1%treadmillgrademostaccuratelyreflectstheenergeticcostofoutdoorrunning.J.SportsSci.14,321–327.

Jurkowski, J.E., Jones,N.L.,Toews,C.J.,Sutton, J.R.,1981.Effectsofmenstrualcycleonblood lactate,O2delivery,andperformanceduringexercise.J.Appl.Physiol.51,1493–1499.

Kaciuba-Uscilko,H.,Grucza,R.,2001.Genderdifferencesinthermoregulation.Curr.Opin.Clin.Nutr.Metab.Care4,533–6.

Keatisuwan,W.,Ohnaka,T.,Tochihara,Y.,1996.PhysiologicalresponsesofmenandwomenduringexerciseinhotenvironemtnswithequivalentWBGT.Appl.Hum.Sci.15,249–258.

Kenefick,R.W.,Cheuvront,S.N.,Sawka,M.N.,2007.Thermoregulatoryfunctionduringthemarathon.Sport.Med.37,312–315.

Kenefick,R.W.,Sawka,M.N.,2007.Heatexhaustionanddehydrationascausesofmarathoncollapse.Sport.Med.37,378–381.

Kenefick,R.W.,Sollanek,K.J.,Charkoudian,N.,Sawka,M.N.,2014.Impactofskintemperatureandhydrationonplasmavolumeresponsesduringexercise.J.Appl.Physiol.117,413–20.

Kenney,W.L.,1985.Areviewofcomparativeresponsesofmenandwomentoheatstress.Environ.Res.37,1–11.

Kenney,W.L.,Hodgson,J.L.,1987.Heattolerance,thermoregulationandageing.Sport.Med.4,446–456.Kenney,W.L., Stanhewicz, A.E., Bruning, R.S., Alexander, L.M., 2014. Blood pressure regualtion III:What

happenswhenonesystemmustservetwomasters:Temperatureandpressureregulation?Eur.J.Appl.Physiol.114,467–479.

Kenny,G.P.,Jay,O.,2013.Thermometry,calorimetry,andmeanbodytemperatureduringheatstress.Compr.Physiol.3,1689–719.

Kenny, G.P., Webb, P., Ducharme, M.B., Reardon, F.D., Jay, O., 2008. Calorimetric measurement ofpostexercisenetheatlossandresidualbodyheatstorage.Med.Sci.SportsExerc.40,1629–36.

Kolka,M.A.,Stephenson,L.A.,1997.Effectoflutealphaseelevationincoretemperatureonforearmbloodflowduringexerciseheat.J.Appl.Physiol.82,1079–1083.

Kolka,M.A., Stephenson, L.A., 1989.Control of sweatingduring thehumanmenstrual cycle. Eur. J.Appl.Physiol.Occup.Physiol.58,890–895.

Kondo,N.,Shibasaki,M.,Aoki,K.,Koga,S.,Inoue,Y.,Crandall,C.G.,2011.Functionofhumaneccrinesweatglandsduringdynamicexerciseandpassiveheatstress.J.Appl.Physiol.90,1877–1881.

Kregel,K.C.,2002.Molecularbiologyof thermoregulation InvitedReview:Heat shockproteins:modifyingfactorsinphysiologicalstressresponsesandacquiredthermotolerance.J.Appl.Physiol.92,2177–2186.

Kresfelder,T.,Claassen,N.,Cronje,M.,2006.Hsp70inductionandhsp70genepolymorphismsasindicatorsofacclimatizationunderhyperthermicconditions.J.Therm.Biol.31,406–415.

Kuennen,M.,Gillum,T.,Dokladny,K.,Bedrick,E.,Schneider,S.,2011.Thermotoleranceandheatacclimation

194

mayshareacommonmechanisminhumans.Am.J.Physiol.Regul.Integr.Comp.Physiol.1,524–533.Kuwahara,T.,Inoue,Y.,Abe,M.,Sato,Y.,Kondo,N.,2005.Effectsofmenstrualcycleandphysicaltrainingon

heatlossresponsesduringdynamicexerciseatmoderateintensityinatemperateenvironment.Am.J.Physiol.Regul.Integr.Comp.Physiol.228,R1347–R1353.

Lakens,D.,2013.Calculatingandreportingeffectsizestofacilitatecumulativescience:apracticalprimerfort-testsandANOVAs.Front.Psychol.4,1–12.

Lebrun,C.M.,McKenzie,D.C.,Prior, J.C., Taunton, J.E., 1995.Effectsofmenstrual cyclephaseonathleticperformance.Med.Sci.SportExerc.27,437–444.

Lee, B.J., Miller, A., James, R.S., Thake, C.D., 2016. Cross acclimation between heat and hypoxia: Heatacclimationimprovescellulartoleranceandexerciseperformanceinacutenormobarichypoxia.Front.Physiol.7,1–15.

Lee,S.H.,Kwon,H.M.,Kim,Y.J.,Lee,K.M.,Kim,M.,Yoon,B.W.,2004.EffectsofHsp70.1geneknockoutonthe mitochondrial apoptotic pathway after focal cerebral ischemia. Stroke 35, 2195–2199.doi:10.1161/01.STR.0000136150.73891.14

Lee, S.M.C., Williams, W.J., Schneider, S.M., 2000. Core temperature measurement during submaximalexercise:Esophageal,rectal,andintestinaltemperatures.Aviat.Sp.Environ.Med.71,939–945.

Lefrant,J.Y.,Muller,L.,deLaCoussaye,J.E.,Benbabaali,M.,Lebris,C.,Zeitoun,N.,Mari,C.,Saïssi,G.,Ripart,J., Eledjam, J.J., 2003. Temperaturemeasurement in intensive care patients: comparisonof urinarybladder, oesophageal, rectal, axillary, and inguinalmethods versus pulmonary artery coremethod.IntensiveCareMed.29,414–8.

Levine, T.R., Hullett, C.R., 2002. Eta Squared, Partial Eta Squared, and Misreporting of Effect Size inCommunicationResearch.Hum.Commun.Res.28,612–625.doi:10.1111/j.1468-2958.2002.tb00828.x

Libert, J.P., Candas, V., Sagot, J.C., Meyer, J.P., Vogt, J.J., Ogawa, T., 1984. Contribution of skin thermalsensitivitiesoflargebodyareastosweatingresponse.Jpn.J.Physiol.34,75–88.

Locke,M.,1997.Thecellularstressresponsetoexercise:roleofstressproteins.Exerc.SportSci.Rev.25,105–136.

Lorenzo,S.,Halliwill,J.R.,Sawka,M.N.,Minson,C.T.,2010.Heatacclimationimprovesexerciseperformance.J.Appl.Physiol.109,1140–1147.

Lorenzo, S.,Minson, C.T., 2010. Heat acclimation improves cutaneous vascular function and sweating intrainedcyclists.J.Appl.Physiol.109,1736–1743.

Loucks,A.B.,Horvath,S.M.,1985.Athleticamenorrhea:areview.Med.Sci.SportExerc.17,56–72.Low, D.A., Vu, A., Brown, M., Davis, S.L., Keller, D.M., Levine, B.D., Crandall, C.G., 2007. Temporal

thermometryfailstotrackbodycoretemperatureduringheatstress.Med.Sci.SportExerc.39,1029–1035.

Low,P..,2004.Evaluationofsudomotorfunction.Clin.Neurophysiol.115,1506–13.Lundvall,J.,Lindgren,P.,1998.F-cellshiftandproteinlossstronglyaffectvalidityofPVreductionsindicated

byHb/Hctandplasmaproteins.J.Appl.Physiol.84,822–829.Magalhães,F.D.C.,Amorim,F.T.,Passos,R.L.F.,Fonseca,M.A.,Oliveira,K.P.M.,Lima,M.R.M.,Guimarães,J.B.,

Ferreira-Júnior,J.B.,Martini,A.R.P.,Lima,N.R.V,Soares,D.D.,Oliveira,E.M.,Rodrigues,L.O.C.,2010.Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-inducedincreaseinintracellularandplasmaHsp72inhumans.CellStressChaperones15,885–95.

Maloyan,A.,Palmon,A.,Horowitz,M.,1999.HeatacclimationincreasesthebasalHSP72levelandaltersitsproductiondynamicsduringheatstress.Am.J.Physiol.276,R1506–15.

Marsh,S.A.,Jenkins,D.G.,2002.Physiologicalresponsestothemenstrualcycle.SportMed.32,601–614.Marshall, H.C., Campbell, S. a., Roberts, C.W.,Nimmo,M. a., 2007. Human physiological and heat shock

protein72adaptationsduringtheinitialphaseofhumid-heatacclimation.J.Therm.Biol.32,341–348.Marshall, H.C., Ferguson, R.A., Nimmo, M.A., 2006. Human resting extracellular heat shock protein 72

concentrationdecreasesduring the initial adaptation toexercise in ahot, humidenvironment. CellStressChaperones11,129–134.

Martin,D.E.,1997.Climaticheatstressandathleticperformance.TrackCoach139.Mauger, A.R.,Metcalfe, A.J., Taylor, L., Castle, P.C., 2013. The efficacy of the self pacedVO2max test to

measuremaximaloxygenuptakeintreadmillrunning.Appl.Physiol.Nutr.Metab.38,1211–1216.Maughan,R.J.,Mcarthur,M.,Shirreffs,S.M.,1996.Influenceofmenstrualstatusonfluidreplacementafter

exerciseinduceddehydrationinhealthyyoungwomen.Br.J.SportMed.30,41–47.Maw,G.J.,Mackenzie,I.L.,Comer,D.A.,Taylor,N.A.,1996.Wholebodyhyperhydrationinendurance-trained

malesdeterminedusingradionuclidedilution.Med.Sci.SportExerc.28,1038–1044.Maw, G.J., Mackenzie, I.L., Taylor, N.., 2000. Can skin temperature manipulation, with minimal core

temperaturechange,influenceplasmavolumeinrestinghumans?Eur.J.Appl.Physiol.81,159–162.

195

McClung,J.P.,Hasday,J.D.,He,J.R.,Montain,S.J.,Cheuvront,S.N.,Sawka,M.N.,Singh,I.S.,2008.Exercise-heatacclimationinhumansaltersbaselinelevelsandexvivoheatinducibilityofHSP72andHSP90inperipheralbloodmononuclearcells.Am.J.Physiol.Regul.Integr.Comp.Physiol.294,R185–191.

Mee, J.A., Gibson, O.R., Doust, J., Maxwell, N.S., 2015. A comparison of males and females’ temporalpatterningtoshort-andlong-termheatacclimation.Scand.J.Med.Sci.Sports25,250–258.

Mekjavic,I.B.,2006.Contributionofthermalandnonthermalfactorstotheregulationofbodytemperatureinhumans.J.Appl.Physiol.100,2065–2072.

Miller,P.B.,Soules,M.R.,1996.TheusefulnessofaurinaryLHkitforovulationpredictionduringmenstrualcyclesofnormalwomen.Am.J.Obstet.Gynecol.87,13–17.

Mitchell, D., Wyndham, C.H., 1969. Comparision of weighting formulas for calculating mean skintemperature.J.Appl.Physiol.26,616.

Montain, S.J., Sawka,M.N., Cadarette, B.S., Quigley,M.D.,McKay, J.M., 1994. Physiological tolerance touncompensable heat stress: effects of exercise intensity, protective clothing, and climate. J. Appl.Physiol.77,216–222.

Mora-rodriguez,R.,Coso,J.D.,Hamouti,N.,Estevez,E.,Ortega,J.F.,2010.Aerobicallytrainedindividualshavegreater increases in rectal temperature than untrained ones during exercise in the heat at similarrelativeintensities.Eur.J.Appl.Physiol.109,973–981.

Moran,D.S.,Eli-Berchoer,L.,Heled,Y.,Mendel,L.,Schocina,M.,Horowitz,M.,2006.Heatintolerance:doesgenetranscriptioncontribute?J.Appl.Physiol.100,1370–1376.

Moran,D.S.,Erlich,T.,Epstein,Y.,2007.Theheattolerancetest:anefficientscreeningtoolforevaluatingsusceptibilitytoheat.J.SportRehabil.16,215–21.

Moran,D.S.,Heled,Y.,Still,L.,Laor,A.,Shapiro,Y.,2004.Assessmentofheattoleranceforpostexertionalheatstrokeindividuals.Med.Sci.Monit.10,CR252–CR257.

Moran,D.S.,Mendal,L.,2002.Coretemperaturemeasurement:methodsandcurrentinsights.Sport.Med.32,879–85.

Moran,D.S.,Shapiro,Y.,Laor,A.,Izraeli,S.,Pandolf,K.B.,1999.Cangenderdifferencesduringexercise-heatstressbeassessedbythephysiologicalstrainindex?Am.J.Physiol.Regul.Integr.Comp.Physiol.276,R1798–R1804.

Moran,D.S.,Shitzer,A.,Pandolf,K.B.,1998.Aphysiologicalstrainindextoevaluateheatstress.Am.J.Physiol.Regul.Integr.Comp.Physiol.275,R129–R134.

Morton,J.P.,Holloway,K.,Woods,P.,Cable,N.T.,Burniston,J.,Evans,L.,Kayani,A.C.,McArdle,A.,2009.Exercise training-induced gender-specific heat shock protein adaptations in human skeletalmuscle.MuscleNerve39,230–233.

Moseley,P.L.,1997.Heatshockproteinsandheatadaptationofthewholeorganism.J.Appl.Physiol.83,1413–1417.

Nadal,E.R.,Bullard,R.W.,Stolwijk,J.A.,1971.Importanceofskintemperatureintheregulationofsweating.J.Appl.Physiol.31,80–87.

Nadal,E.R.,Cafarelli,E.,Roberts,M.F.,Wenger,C.B.,1979.Circulatoryregulationduringexerciseindifferentambianttemperatures.J.Appl.Physiol.46,430–437.

Nakamura,K.,Matsumura,K.,Hübschle,T.,Nakamura,Y.,Hioki,H.,Fujiyama,F.,Boldogköi,Z.,König,M.,Thiel,H.-J.,Gerstberger,R., Kobayashi, S., Kaneko, T., 2004. Identificationof sympatheticpremotorneuronsinmedullaryrapheregionsmediatingfeverandotherthermoregulatoryfunctions.J.Neurosci.24,5370–80.

Nakayama,T.,Suzuki,M., Ishizuka,N.,1975.Actionofprogesteroneonpreopticthermosensitiveneurons[abstract].Nature258.

Neal,R.A.,Corbett,J.,Massey,H.C.,Tipton,M.J.,2015.Effectofshort-termheatacclimationwithpermissivedehydration on thermoregulation and temperate exercise performance. Scand. J.Med. Sci. Sports.doi:10.1111/sms.12526

Nelson,M.D.,Haykowsky,M.J.,Petersen,S.R.,Delorey,D.S.,Stickland,M.K.,Cheng-baron,J.,Thompson,R.B.,2010.Aerobicfitnessdoesnotinfluencethebiventricularresponsetowholebodypassiveheatstress.J.Appl.Physiol.109,1545–1551.

Nielsen,B.,1996.OlympicsinAtlanta:afightagainstphysics.Med.Sci.SportExerc.28,665–668.Nielsen,B.Y.B.,Hales,J.R.S.,Strange,S.,Juel,N.,1993.Humancirculatoryandthermoregulatoryadaptations

withheatacclimationandexerciseinahot,dryenvironment.J.Physiol.460,467–485.Nose,H.,Mack,G..,Shi,X.R.,Nadel,E.R.,1988.Shiftinbodyfluidcompartmentsafterdehydrationinhumans.

J.Appl.Physiol.65,318–324.Nose,H.,Mack,G.W.,Shi,X.R.,Morimoto,K.,Nadel,E.R.,1990.Effectofsalineinfusionduringexerciseon

thermalandcirculatoryregulations.J.Appl.Physiol.69,609–616.

196

Nunneley,S.,1978.Physiologicalresponsesofwomentothermalstress:Areview.Med.Sci.Sports10,250–255.

Nybo,L.,Nielsen,B.,2001.Hyperthermiaandcentralfatigueduringprolongedexerciseinhumans.J.Appl.Physiol.91,1055–60.

Nybo,L.,Rasmussen,P.,Sawka,M.N.,2014.Performanceintheheat-physiologicalfactorsofimportanceforhyperthermia-inducedfatigue.Compr.Physiol.4,657–89.

Nybo,L.,Secher,N.H.,Nielsen,B.,2002.Inadequateheatreleasefromthehumanbrainduringprolongedexercisewithhyperthermia.J.Physiol.545,697–704.

Ogita,H.,Node,K.,Kitakaze,M.,2003.Theroleofestrogenandestrogen-relateddrugs incardiovasculardiseases.Curr.DrugMetab.4,497–504.

Pandolf,K.B.,1998.Timecourseofheatacclimationanditsdecay.Int.J.SportsMed.19,S157–S160.Paroo,Z.,Dipchand,E.S.,Noble,E.G.,2002.EstrogenattenuatespostexerciseHSP70expressioninskeletal

muscle.Am.J.Physiol.CellPhysiol.282,C245–51.Patterson, M.J., Stocks, J.M., Taylor, N. a S., 2014. Whole-body fluid distribution in humans during

dehydration and recovery, before and after humid-heat acclimation induced using controlledhyperthermia.ActaPhysiol.210,899–912.

Patterson,M.J.,Stocks,J.M.,Taylor,N.aS.,2004a.Humidheatacclimationdoesnotelicitapreferentialsweatredistributiontowardthelimbs.Am.J.Physiol.Regul.Integr.Comp.Physiol.286,R512–R518.

Patterson,M.J.,Stocks,J.M.,Taylor,N.aS.,2004b.Sustainedandgeneralizedextracellularfluidexpansionfollowingheatacclimation.J.Physiol.559,327–34.

Périard,J.D.,2013.Cardiovasculardeterminantsinvolvedinpacingunderheatstress.Sport.Med.43,643–5.Périard,J.D.,Cramer,M.N.,Chapman,P.G.,Caillaud,C.,Thompson,M.W.,2011.Cardiovascularstrainimpairs

prolongedself-pacedexerciseintheheat.Exp.Physiol.96,134–44.Périard,J.D.,Jay,O.,Alonso,J.M.,Coutts,A.J.,Flouris,A.D.,González-Alonso,J.,Hausswirth,C.,Lee,J.K.W.,

Nassis,G.P.,Nybo,L.,Pluim,B.M.,Roelands,B.,Sawka,M.N.,Wingo, J.,Racinais,S.,2016.Author’sReplytoBrocherieandMillet:‘IstheWet-BulbGlobeTemperature(WGBT)Index.Sport.Med.46,139–141.

Périard, J.D., Racinais, S., Sawka, M.N., 2015. Adaptations and mechanisms of human heat acclimation:Applicationsforcompetitiveathletesandsports.Scand.J.Med.Sci.Sports25,20–38.

Périard, J.D., Ruell, P., Caillaud, C., Thompson,M.W., 2012. Plasma Hsp72 (HSPA1A) and Hsp27 (HSPB1)expressionunderheatstress:influenceofexerciseintensity.CellStressChaperones17,375–83.

Pockley,G.,2003.Heatshockproteinsasregulatorsoftheimmuneresponse.Lancet362,469–76.Poirier,M.P.,Gagnon,D.,Friesen,B.J.,Hardcastle,S.G.,Kenny,G.P.,2015.Whole-bodyheatexchangeduring

heatacclimationanditsdecay.Med.Sci.SportExerc.47,390–400.Prud’Homme, D., Bouchard, C., Leblanc, C., Landry, F., Fontaine, E., 1984. Sensitivity ofmaximal aerobic

powertotrainingisgenotypedependent.Med.Sci.SportExerc.18,489–493.Racinais,S.,Alonso,J.M.,Coutts,a.J.,Flouris,a.D.,Girard,O.,González-Alonso,J.,Hausswirth,C.,Jay,O.,

Lee, J.K.W.,Mitchell,N.,Nassis,G.P.,Nybo, L., Pluim,B.M.,Roelands,B., Sawka,M.N.,Wingo, J.E.,Périard,J.D.,2015.Consensusrecommendationsontrainingandcompetingintheheat.Scand.J.Med.Sci.Sports25,6–19.

Racinais,S.,Mohr,M.,Buchheit,M.,Voss,S.C.,Gaoua,N.,Grantham,J.,Nybo,L.,2012.Individualresponsestoshort-termheatacclimatisationaspredictorsoffootballperformanceinahot,dryenvironment.Br.J.SportsMed.46,810–5.

Racinais,S.,Périard,J.D.,Karlsen,A.,Nybo,L.,2015.Effectofheatandheatacclimatizationoncyclingtimetrialperformanceandpacing.Med.Sci.SportsExerc.47,601–6.

Ramanathan,N.L.,1964.Anewweightingsystemformeansurfacetemperatureofthehumanbody.J.Appl.Physiol.19,531–533.

Randall,W.C.,1946.Quantificationandregionaldistributionofsweatglandsinman.J.Clin.Invest.25,761–767.

Rasmussen, P.,Nybo, L., Volianitis, S.,Møller, K., Secher,N.H.,Gjedde,A., 2010. Cerebral oxygenation isreducedduringhyperthermicexerciseinhumans.ActaPhysiol.199,63–70.

Regan,J.M.,Macfarlane,D.J.,Taylor,N.A.S.,1996.Anevaluationoftheroleofskintemperatureduringheatadaptation.ActaPhysiol.Scand.158,365–375.

Regan, J.M.,Macfarlane,D.J.,Taylor,N.A.S.,1994.Adaptation toheat:differences inducedby isothermalstrain following heat acclimation and thermoneutral exercise, in: International Conference onEnvironmentalErgonomics.pp.45–46.

RichardDavison,R.C.,Wooles,A.L.,2007.Cycling,in:Winter,E.,Jones,A.,RichardDavison,R.C.,Bromley,P.D.,Mercer,T.H.(Eds.),SportandExercisePhysiologyTestingGuidlines.RoutledgeTaylorandFrancis

197

Group,LondonandNewYork,pp.160–164.Richards,R.,Richards,D.,1984.ExertioninducedheatexhaustionandothermedicalaspectsoftheCity-to-

Surffunruns,1978-1984.Med.J.Aust.141,799–805.Roberts,M.F.,Wenger,C.B.,Stolwijk,J.A.,Nadal,E.R.,1977.Skinbloodflowandsweatingchangesfollowing

exercisetrainingandheatacclimation.J.Appl.Physiol.43,133–137.Roberts,W.O., 2010. Determining a “do not start” temperature for amarathon on the basis of adverse

outcomes.Med.Sci.SportExerc.42,226–232.Roberts,W.O.,2006.exertionalheatstrokeduringacoolweathermarathon:acasestudy.Med.Sci.Sport

Exerc.38,1197–1203.Roberts,W.O.,2000.A12-yrprofileofmedicalinjuryandillnessfortheTwinCitiesMarathon.Med.Sci.Sport

Exerc.32,1549–1555.Robertson,B.,Walter,E.,2010.“Coolrunnings”:heatstrokeincoolconditions.Emerg.Med.J.27,387–388.Robinson,J.,Charlton,J.,Seal,R.,Spady,D.,Joffres,M.R.,1998.Oesophageal,rectal,axillary,tympanic,and

pulmonaryarterytemperaturesduringcardiacsurgery.Can.J.Anaesth.45,317–323.Robinson,S.,1963.Temperatureregulationinexercise.Pediatrics32,691–702.Rowell,L.B.,Kraning,K.K.,Kennedy,J.W.,Evans,T.O.,1967.Centralcirculatoryresponsestoworkindryheat

beforeandafteracclimatization.J.Appl.Physiol.22,509–518.Ruell, P.A., Simar,D., Périard, J.D., Best, S., Caillaud, C., Thompson,M.W., 2014. Plasmaand lymphocyte

Hsp72responsestoexerciseinathleteswithpriorexertionalheatillness.AminoAcids46,1491–9.Saat, M., Sirisinghe, R.G., Singh, R., Tochihara, Y., 2005. Effects of short-term exercise in the heat on

thermoregulation, blood parameters, sweat secretion and sweat composition of tropic-dwellingsubjects.J.Physiol.Anthropol.Appl.HumanSci.24,541–549.

Saltin,B.,Hermansen,L.,1966.Esophageal,rectalandmuscletemperatureduringexercise.J.Appl.Physiol.21,1757–1762.

Sandström,M.E.,Siegler,J.C.,Lovell,R.J.,Madden,L.a,McNaughton,L.,2008.Theeffectof15consecutivedaysofheat-exerciseacclimationonheatshockprotein70.CellStressChaperones13,169–75.

Sato,F.,Owen,M.,Matthes,R.,Sato,K.,Gisolfi,C.V,1990.Functionalandmorphologicalglandwithheatacclimationchangesintheeccrinesweatglandwithheatacclimation.J.Appl.Physiol.69,232–236.

Sato,K.,Dobson,R.L.,1970.Regionalandindividualvariationsinthefunctionofthehumaneccrinesweatgland.J.Investig.Dematology54,443–449.

Sato,K.,Dobson,R.L.,Mali,J.W.H.,1971.Enxymaticbasisfortheactivetransportofsodiumintheeccrinesweatgland.J.Invest.Dermatol.57,10–16.

Sato,K.,Kang,W.H.,Saga,K.,Sata,K.T.,1989.Biologyofsweatglandsandtheirdisorders.I.Normalsweatglandfunction.J.Am.Acad.Dermotology20,537–563.

Sato,K.,Sato,F.,1983.Individualvariationsinstructureandfunctionofhumaneccrinesweatgland.Am.J.Physiol.Regul.Integr.Comp.Physiol.245,R203–R208.

Sawka,M.N., Burke, L.M., Eichner, E.R., Maughan, R.J., Montain, S.J., Stachenfeld, N.S., 2007. AmericanCollegeofSportsMedicinepositionstand.Exerciseandfluidreplacement.Med.Sci.SportExerc.39,377–390.

Sawka,M.N.,Coyle,E.F.,1999.Influenceofbodywaterandbloodvolumeonthermoregulatoryandexerciseperformanceintheheat.Exerc.SportSci.Rev.27,167–218.

Sawka, M.N., Gonzalez, R.R., Young, A.J., Dennis, R.C., Valarie, R.C., Pandolf, K.B., 1989. Control ofthermoregulatoryduringexerciseintheheat.Am.J.Physiol.Regul.Integr.Comp.Physiol.26,311–316.

Sawka,M.N.,Leon,L.R.,Montain,S.J.,Sonna,L.A.,2011a.Integratedphysiologicalmechanismsofexerciseperformance,adaptation,andmaladaptationtoheatstress.Compr.Physiol.1,1883–1928.

Sawka,M.N.,Toner,M.M.,Francesconi,R.P.,Pandolf,K.B.,1983.Hypohydrationandexercise:effectsofheatacclimation,genderandenvironment.J.Appl.Physiol.55,1147–1153.

Sawka,M.N.,Wenger,C.B.,Pandolf,K.B.,2011b.Thermoregulatoryresponsestoacuteexerciseheatstressandheatacclimation.Compr.Physiol.157–185.

Sawka,M.N.,Wenger,C.B.,Pandolf,K.B.,1993.Humanresponsestoexerciseheatstress.Schaible, T.F., Scheuer, J., 1979. Effects of physical training by running or swimming on ventricular

performanceofrathearts.J.Appl.Physiol.Repiratory,Environ.Exerc.Physiol.46,854–60.Scherr, J.,Wolfarth, B., Christle, J.W., Pressler, A.,Wagenpfeil, S., Halle,M., 2013. Associations between

Borg’s rating of perceived exertion and physiological measures of exercise intensity. Eur. J. Appl.Physiol.113,147–55.

Schlader,Z.J.,Raman,A.,Morton,R.H.,Stannard,S.R.,Mündel,T.,2011a.Exercisemodalitymodulatesbodytemperatureregulationduringexerciseinuncompensableheatstress.Eur.J.Appl.Physiol.111,757–66.

198

Schlader,Z.J.,Simmons,S.E.,Stannard,S.R.,Mündel,T.,2011b.Physiology&Behavior:Theindependentrolesoftemperatureandthermalperceptioninthecontrolofhumanthermoregulatorybehavior.Physiol.Behav.103,217–224.

Schmittgen,T.D.,Livak,K.J.,2008.Analyzingreal-timePCRdatabythecomparativeCTmethod.Nat.Protoc.3,1101–1108.

Schutte,P.,2010.Heatstressmanagementinhotmines,in:Brune,J.P.(Ed.),ExtractingtheScience:ACenturyofMiningResearch.USA,pp.30–34.

Scoon,G.S.M.,Hopkins,W.G.,Mayhew,S.,Cotter,J.D.,2007.Effectofpost-exercisesaunabathingontheenduranceperformanceofcompetitivemalerunners.J.Sci.Med.Sport10,259–262.

Senay,L.C.,1973.Bodyfluidandtemperatureresponsesofheatexposedwomenbeforeandafterovulationwithandwithoutrehydration.J.Physiol.232,209–219.

Senay, L.C.,Mitchell, D.,Wyndham, C.H., 1976. Acclimatization in a hot, humid environment: body fluidadjustments.J.Appl.Physiol.40,786–796.

Senf,S.M.,Howard,T.M.,Ahn,B.,Ferreira,L.F., Judge,A.R.,2013.Lossofthe InducibleHsp70DelaystheInflammatoryResponsetoSkeletalMuscleInjuryandSeverelyImpairsMuscleRegeneration.PLoSOne8.

Shapiro,Y.,Hubbard,R.W.,Kimbrough,C.M.,Pandolf,K.B.,1981.Physiologicalandhematologicalresponsestosummerandwinterdryheatacclimation.J.Appl.Physiol.50,792–798.

Shapiro,Y.,Magazanik,A.,Udassin,R.,Ben-Baruch,G.,Shvartz,E.,Shoenfeld,Y.,1979.Heatintoleranceinformerheatstrokepatients.Ann.Intern.Med.90,913–916.

Shibasaki,M.,Wilson,T.E.,Crandall,C.G.,2006.Neuralcontrolandmechanismsofeccrinesweatingduringheatstressandexercise.J.Appl.Physiol.100,1692–1701.

Shinohara,T.,Takahashi,N.,Ooie,T., Ichinose,M.,Hara,M.,Yonemochi,H., Saikawa,T.,Yoshimatsu,H.,2004. Estrogen inhibits hyperthermia-induced expression of heat-shock protein 72 andcardioprotectionagainstischemia/reperfusioninjuryinfemaleratheart.J.Mol.Cell.Cardiol.37,1053–1061.

Shvartz, E., Bhattacharya, A., Sperinde, S.J., Brock, P.J., Sciaraffa, D., Van Beaumont,W., 1979. Sweatingresponsesduringandmoderateconditioning.J.Appl.Physiol.46,675–680.

Silva,N.L.,Boulant, J.a,1986.Effectsof testosterone,estradiol,and temperatureonneurons inpreoptictissueslices.Am.J.Physiol.250,R625–R632.

Silver,J.T.,Noble,E.G.,2012.Regulationofsurvivalgenehsp70.CellStressChaperones17,1–9.Skidmore,R.,Gutierrez,J.a,GuerrieroJr,V.,Kregel,K.C.,1995.HSP70inductionduringexerciseandheat

stressinrats:roleofinternaltemperature.Am.J.Physiol.268,R92–R97.Smith,A.,Crabtree,D.,Bilzon,J.,Walsh,N.,2010.ThevalidityofwirelessiButtonsandthermistorsforhuman

skintemperaturemeasurement.Physiol.Meas.31,95–114.Stacey,M.J.,Parsons,I.T.,Woods,D.R.,Taylor,P.N.,Ross,D.,JBrett,S.,2015.Susceptibilitytoexertionalheat

illnessandhospitalisationriskinUKmilitarypersonnel.Br.Med.J.OpenSportExerc.Med.1,e000055.Stachenfeld, N.S., Pietro, L.D.I., Palter, S.F., Nadel, E.R., Nina, S., Dipietro, L., Steven, F., 1998. Estrogen

influencesosmoticsecretionofAVPandbodywaterbalanceinpostmenopausalwomen.Am.J.Physiol.Regul.Integr.Comp.Physiol.274,187–195.

Stachenfeld,N.S.,Silva,C.,Keefe,D.L.,2000.Estrogenmodifiesthetemperatureeffectsofprogesterone.J.Appl.Physiol.88,1643–1649.

Stachenfeld,N.S.,Silva,C.,Keefe,D.L.,Kokoszka,C.A.,Nadel,E.R.,1999.Effectsoforalcontraceptivesonbodyfluidregulation.J.Appl.Physiol.87,1016–1025.

Stachenfeld, N.S., Taylor, H.S., 2014. Challenges and methodology for testing young healthy women inphysiologicalstudies.Am.J.Endocrinol.Metab.06519,849–853.

Stanley,J.,Halliday,A.,D’Auria,S.,Buchheit,M.,Leicht,A.S.,2015.Effectofsauna-basedheatacclimationonplasmavolumeandheartratevariability.Eur.J.Appl.Physiol.115,785–94.

Stapleton,J.M.,Larose,J.,Simpson,C.,Flouris,A.D.,Sigal,R.J.,Kenny,G.P.,2014.Doolderadultsexperiencegreaterthermalstrainduringheatwaves?Appl.Physiol.Nutr.Metab.39,292–8.

Stephenson, L.A., Kolka, M.A., 1999. Esophageal temperature threshold for sweating decreases beforeovulationinpremenopausalwomen.J.Appl.Physiol.86,22–28.

Stephenson,L.A.,Kolka,M.A.,1993.ThermoregulationinWomen.Exerc.SportSci.Rev.21,231–262.Stephenson,L.A.,Kolka,M.A.,1985.Menstrualcyclephaseandtimeofdayalterreferencesignalcontrolling

armbloodflowandsweating.Am.J.Physiol.Regul.Integr.Comp.Physiol.249,R186–R191.Sunderland,C.,Morris,J.G.,Nevill,M.E.,2008.Aheatacclimationprotocolforteamsports.Br.J.SportsMed.

42,327–333.Syrop,C.H.,Hammond,M.G.,1987.Diurnalvariationsinmidlutealserumprogesteronemeasurements.Fertil.

199

Steril.47,67–70.Takamata, A., Yoshida, T., Nishida, N., Morimoto, T., 2001. Relationship of osmotic inhibition in

thermoregulatoryresponsesandsweatsodiumconcentrationinhumans.Am.J.Physiol.Regul.Integr.Comp.Physiol.280,623–629.

Takeno,Y.,Kamijo,Y.I.,Nose,H.,2001.Thermoregulatoryandaerobicchangesafterendurancetraininginahypobarichypoxicandwarmenvironment.J.Appl.Physiol.91,1520–8.

Tankersley, C.G., Nicholas, W.C., Deaver, D.R., Mikita, D., Kenney, W.L., 1992. Estrogen replacement inmiddle-agedwomen :thermoregulatoryresponsestoexerciseintheheat.J.Appl.Physiol.73,1238–1245.

Taylor,L.,Midgley,A.W.,Chrismas,B.,Hilman,A.R.,Madden,L.a,Vince,R.V,McNaughton,L.R.,2011.DailyhypoxiaincreasesbasalmonocyteHSP72expressioninhealthyhumansubjects.AminoAcids40,393–401.

Taylor,N.A.S.,2000.Principlesandpracticesofheatadaptation.J.Hum.EnvionmentalSyst.4,11–22.Taylor,N.A.S.,Cotter,J.D.,2006.Heatadaptation:Guidlinesfortheoptimisationofhumanperformance.Int.

J.SportsMed.7,33–57.Taylor,N.A.S.,Tipton,M.J.,Kenny,G.P.,2014.Considerationsforthemeasurementofcore,skinandmean

bodytemperatures.J.Therm.Biol.46,72–101.doi:10.1016/j.jtherbio.2014.10.006Taylor,N.S.,2014.Humanheatadaptation.Compr.Physiol.4,325–65.Taylor,W.F.,Johnson,J.M.,O’Leary,D.,Park,M.K.,1984.Effectofhighlocaltemperatureonreflexcutaneous

vasodilation.J.Appl.Physiol.57,191–196.Tenaglia,S.a,McLellan,T.M.,Klentrou,P.P.,1999.Influenceofmenstrualcycleandoralcontraceptiveson

tolerancetouncompensableheatstress.Eur.J.Appl.Physiol.Occup.Physiol.80,76–83.Tetievsky,A.,Cohen,O.,Eli-Berchoer,L.,Gerstenblith,G.,Stern,M.D.,Wapinski,I.,Friedman,N.,Horowitz,

M.,2008.Physiologicalandmolecularevidenceofheatacclimationmemory:a lesson fromthermalresponsesandischemiccross-toleranceintheheart.Physiol.Genomics34,78–87.

Toner,M.M.,Drolet,L.L.,Pandolf,K.B.,1986.Perceptualandphysiologicalresponsesduringexerciseincoolandcoldwater.Percept.Mot.Skills62,211–220.

Tucker,R.,Marle,T.,Lambert,E.V,Noakes,T.D.,2006.Therateofheatstoragemediatesananticipatoryreductioninexerciseintensityduringcyclingatafixedratingofperceivedexertion.J.Physiol.574,905–915.

Tucker, R., Rauch, L., Harley, Y.X.R., Noakes, T.D., 2004. Impaired exercise performance in the heat isassociatedwithananticipatoryreductioninskeletalmusclerecruitment.Eur.J.Physiol.448,422–30.

Turk,J.,Thomas,I.R.,1975.Artificialacclimatizationtoheat.Ann.Occup.Hygeine17,271–278.Tuttle, J. a,Castle,P.C.,Metcalfe,A.J.,Midgley,A.W.,Taylor, L., Lewis,M.P.,2015.Downhill runningand

exerciseinhotenvironmentsincreaseleukocyteHsp72(HSPA1A)andHsp90α(HSPC1)genetranscripts.J.Appl.Physiol.118,996–1005.

Vincent,W.J.,1995.StatisticsinKinesiology.HumanKinetics.Webb,P.,1995.ThePhysiologyofheatregualtion.Am.J.Physiol.Regul.Integr.Comp.Physiol.268,R838–

R850.Weller,A.S.,Linnane,D.M.,Jonkman,A..,Daanen,H.A.M.,2007.Quantificationofthedecayandre-induction

ofheatacclimationindry-heatfollowing12and26dayswithoutexposuretoheatstress.Eur.J.Appl.Physiol.102,57–66.

Wells,C.L.,Horvath,S.M.,1974.Responsestoexerciseinahotenvironmentasrelatedtothemenstrualcycle.J.Appl.Physiol.36,299–302.

Whipp,B.J.,Wasserman,K.,1972.Oxygenuptakekinetics forvarious intensitiesofconstant-loadwork. J.Appl.Physiol.33,351–356.

Williams,T.J.,Krahenbuhl,G.S.,1997.Menstrualcyclephaseandrunningeconomy.Med.Sci.SportExerc.29,1609–1618.

Wilson,T.E.,Brothers,R.M.,Tollund,C.,Dawson,E.a,Nissen,P.,Yoshiga,C.C.,Jons,C.,Secher,N.H.,Crandall,C.G.,2009.EffectofthermalstressonFrank-Starlingrelationsinhumans.J.Physiol.587,3383–92.

Wilson,T.E.,Cui,J.,Zhang,R.,Crandall,C.G.,2006.Heatstressreducescerebralbloodvelocityandmarkedlyimpairsorthostatictoleranceinhumans.Am.J.Physiol.Regul.Integr.Comp.Physiol.291,R1443–8.

Wingo,J.E.,Low,D.a,Keller,D.M.,Brothers,R.M.,Shibasaki,M.,Crandall,C.G.,2010.Skinbloodflowandlocal temperature independentlymodify sweat rate during passive heat stress in humans. J. Appl.Physiol.109,1301–6.

Wyndham,C.H.,1973.Thephysiologyofexerciseunderheatstress.Annu.Rev.Physiol.35,193–220.Wyndham,C.H.,1951.Effectofacclimatizationoncirculatoryresponsestohighenvironmentaltemperatures.

J.Appl.Physiol.4,383–395.

200

Wyndham,C.H.,Benade,aJ.,Williams,C.G.,Strydom,N.B.,Goldin,A.,Heyns,aJ.,1968.Changesincentralcirculationandbodyfluidspacesduringacclimatizationtoheat.J.Appl.Physiol.25,586–593.

Wyndham,C.H., Rogers,G.G., Senay, L.C.,Mitchell,D., 1976.Acclimization in ahot, humidenvironment:cardiovascularadjustments.J.Appl.Physiol.40,779–785.

Yaglou,C.P.,Minard,D.,1957.Controlofheatcasualtiesatmilitarytrainingcenters.AMAArch.Ind.Heal.16,302–316.

Yamada,P.M.,Amorim,F.T.,Moseley,P.,Robergs,R.,Schneider,S.M.,2007.Effectofheatacclimationonheatshockprotein72andinterleukin-10inhumans.J.Appl.Physiol.103,1196–204.

Yamazaki,F.,Hamasaki,K.,2003.Heatacclimationincreasesskinvasodilationandsweatingbutnotcardiacbaroreflexresponsesinheat-stressedhumans.J.Appl.Physiol.95,1567–1574.

Yoda,T.,Crawshaw,L.I.,Nakamura,M.,Saito,K.,Konishi,A.,Nagashima,K.,Uchida,S.,Kanosue,K.,2005.Effectsofalcoholonthermoregulationduringmildheatexposureinhumans.Alcohol36,195–200.

Zurawlew,M.J.,Walsh,N.P.,Fortes,M.B.,Potter,C.,2015.Post-exercisehotwaterimmersioninducesheatacclimation and improves endurance exercise performance in the heat. Scand. J. Med. Sci. Sport.doi:10.1111/sms.12638

201

Appendices

Informedconsent

INFORMEDCONSENTFORM

Declaration

I herebyvolunteertotakepartinthisresearchprojectwhichinvestigates

Theprincipalinvestigatorhasexplainedtomysatisfactionthepurposeoftheexperimentandthepossiblerisksinvolved.IhavehadtheprinciplesandtheprocedureexplainedtomeandIhavealsoreadtheparticipantinformationsheet.Iunderstandtheprinciplesandproceduresfully.

IamawarethatIwillberequiredto:

Havemyaerobiccapacityandbodycompositionmeasured

Performcyclingexerciseforupto90-mininhotconditionsonoccasions

Havecoretemperaturemonitoredviarectalthermometryonmultipleoccasions.

Iunderstandhowthedatacollectedwillbeused,andthatanyconfidentialinformationwillnormallybeseenonlybytheresearchersandwillnotberevealedtoanyoneelse.

Iunderstandthat Iamfreetowithdrawfromthe investigationatanytimeandthat Iamundernoobligationtogivereasonsforwithdrawalortoattendagainforexperimentation.

IagreethatshouldIwithdrawfromthestudy,thedatacollecteduptothatpointmaybeusedbytheresearcherforthepurposesdescribedintheinformationsheet.

Iunderstandthattheresultsofthestudycanbemadeknowntome.

Furthermore, if I am a student, I am aware that taking part, or not taking part in this experiment, will neither bedetrimentalto,norfurthermypositionasastudent.

Iunderstandtoobeythelaboratory/studyregulationsandtheinstructionsoftheinvestigatorsregardingsafety,subjectonlytomyrighttowithdrawdeclaredabove.

SignatureoftheSubject Date

SignatureoftheInvestigator Date

202

Medicalquestionnaire

MEDICALQUESTIONNAIRE

Name Age

Areyouingoodhealth? Yes No

IfNo,pleaseprovidegivedetails

Howoftendoyoucurrentlyparticipateinvigorousphysicalactivity?

<Oncepermonth

Oncepermonth

2-3timesperweek

4-5timesperweek

5+timesperweek

Haveyousufferedfromaseriousillnessoraccident? Yes No

Ifyes,pleasegiveparticulars:

Doyousuffer,orhaveyoueversufferedfrom:

RespiratoryProblems(e.g.Asthma,Bronchitis?) Yes No

Diabetes? Yes No

Epilepsy? Yes No

High/LowBloodPressure Yes No

Cardiovascularproblems Yes No

Areyoucurrentlytakingmedicationordietarysupplements? Yes No

Ifyes,pleasegivedetails:

Inthelast3months,haveyouconsultedyourGPforanycondition? Yes No

Ifyes,pleasegiveparticulars

Areyoucurrently/recentlytakingpartinanyotherlaboratoryexperiments? Yes No

203

PLEASEREADTHEFOLLOWINGCAREFULLY

Personswillbeconsideredunfittoparticipateinthestudyifthey:

Areunsureofthetestprotocolandthepossiblerisksanddiscomfortsdesignatedonthesubjectinformationsheet;

Theanswersgivenonthemedicalquestionnaireorinformedconsentformdonotmeettherequiredcriteria;

Havesuspendedtrainingduetoaninjury

Haveaknownhistoryofmedicaldisordersincluding,highbloodpressureandheartorlungdisease

Havebeenverified,ordocumentedashavinganybloodcarriedinfections(Hepatitis,HIV),

Havehadapreviousincidenceofhyperthermia,heatstrokeorotherheat-relatedillness

Havesymptomsofnausea,lightheadednessoranaphylacticshocktoneedles,probesorothermedicaltypeequipment

Havechronicoracutesymptomsofgastrointestinalbacterialinfections

Haveahistoryofinfectiousdiseases

DECLARATION

I herebyvolunteertobeaparticipantinexperimentsandinvestigationsconductedduringtheperiodcommencing

MyrepliestotheabovequestionsarecorrecttothebestofmybeliefandIunderstandthattheywillbetreatedwiththestrictestconfidence.Theexperimenterhasfullyinformedmeof,andIhaveunderstood,thepurposeoftheexperimentandpossiblerisksinvolved.

IunderstandthatImaywithdrawfromtheexperimentatanytimeandthatIamundernoobligationtogivereasonsforwithdrawalortoattendagainforexperimentation.

Iundertaketoobeythelaboratory/studyregulationsandtheinstructionsoftheexperimenterregardingsafety,subjectonlytomyrighttowithdrawdeclaredabove.

SignatureoftheSubject Date

SignatureoftheInvestigator Date

204

Menstrualcyclequestionnaire

Werequireinformationregardingyourmenstrualcycletiming.Wherepossibletestingwillbescheduledinthefollicularphaseofthemenstrualcycle(Day3–10).

Wemayrequireavenousbloodsampletoquantifyconcentrationsof17-ßestradiolandprogesterone.Thiswillenableustoconfirmwhetherthetestingwasconductedduringthecorrectphaseofthemenstrualcycle.

Itisessentialthatyoutakeyourtimetocompletethisquestionnairetoensureaccuracyoftheinformationprovided.

Whatistheaveragelengthofyourcycle?

Areyourmenstrualcyclesregular?

Pleaseprovidethestartdateofyouprevioustwomenstrualperiods?

Werethesetwocyclestypicalofyouraveragecycle?

Doyoutakeoralcontraceptiveorusehormonereplacement?

Ifsopleaseprovidedetails:

Pleaseprovideanyadditionalnoteswhichmayberelevant:

205

Calculationof65%VbO2peak

PeakVCO2=2.618L.min-1

VCO2duringcyclingincrementalVCO2peaktest

Calculationof65%peakVCO2

2.618*0.65=1.7017L.min-1

Equationofastraightlinetocalculationpowerat65%VCO2peak

x=(1.7017-0.9176)/0.0084

=93.4watts

y=0.0084x+0.91761.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

50 100 150 200 250

VO2(L.m

in⁻¹)

Power(watts)

Documents

HEAT TOLERANCE AND ACCLIMATION IN FEMALE ATHLETES