Draft - University of Toronto T-Space€¦ · Draft 2 ABSTRACT Introduction: Protein (PRO) combined with a carbohydrate (CHO) beverage may have an ergogenic effect on endurance performance

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Ingestion of carbohydrate or carbohydrate plus protein

does not enhance performance during endurance exercise: a randomized cross-over placebo-controlled clinical trial.

Journal: Applied Physiology, Nutrition, and Metabolism

Manuscript ID apnm-2017-0835.R1

Manuscript Type: Article

Date Submitted by the Author: 04-Mar-2018

Complete List of Authors: Finger, Débora; Universidade Federal do Rio Grande do Sul, Physical Education Lanferdini, Fábio ; Universidade Federal do Rio Grande do Sul, Physical Education Farinha, Juliano; Universidade Federal do Rio Grande do Sul, Brusco, Clarissa; Universidade Federal do Rio Grande do Sul, Physical Education Helal, Lucas; Universidade Federal do Rio Grande do Sul Boeno, Francesco; Universidade Federal do Rio Grande do Sul, Program of Human Movement Sciences, Faculty of Physical Education, Physiotherapy and Dance (ESEFID) Cadore, Eduardo Lusa; Federal University of Rio Grande do Sul, Exercise Research Laboratory Pinto, Ronei; Federal University of Rio Grande do Sul, Physical Education

Keyword: running < sports, cycling < sports, Dietary Supplements, sports performance < sports, Sports Nutrition Sciences

Is the invited manuscript for consideration in a Special

Issue? : N/A

https://mc06.manuscriptcentral.com/apnm-pubs

Applied Physiology, Nutrition, and Metabolism

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1

Ingestion of carbohydrate or carbohydrate plus protein does not enhance

performance during endurance exercise: a randomized cross-over placebo-

controlled clinical trial.

1Débora Finger; 1,2Fábio Juner Lanferdini; 1Juliano Boufleur Farinha; 1Clarissa Müller

Brusco; 3Lucas Helal; 1Francesco Pinto Boeno; 1Eduardo Lusa Cadore, 1Ronei Silveira

Pinto

1 Exercise Research Laboratory - School of Physical Education, Physiotherapy and

Dance - Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

2 Physical Education Course - Universidade Regional Integrada do Alto Uruguai e das

Missões, Santo Ângelo, RS, Brazil.

3 Exercise Pathophysiology Laboratory, School of Medicine, Graduate Program in

Cardiology and Cardiovascular Sciences, Hospital de Clinicas de Porto Alegre,

Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil.

Corresponding author:

Ronei Silveira Pinto

Exercise Research Laboratory

School of Physical Education, Physiotherapy and Dance - Universidade Federal do Rio

Grande do Sul Porto Alegre – RS / Brazil

Rua Felizardo, 750 – Bairro Jardim Botânico

CEP: 90690-200

E-mail: [email protected]

Telephone: +55 51 33085894

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ABSTRACT

Introduction: Protein (PRO) combined with a carbohydrate (CHO) beverage may have

an ergogenic effect on endurance performance. However, evidence regarding its

efficacy on similar conditions to athletes’ race day is still lacking. Objective: To

compare the effect of three different nutritional supplementation strategies on

performance and muscle recovery in a duathlon protocol. Methods:, 13 male athletes

(29.7 ± 7.7 years) participated in three simulated Olympic-distance duathlons under

three different, randomly assigned, supplementation regimens: carbohydrate drink

(CHO, 75 g); isocaloric CHO plus protein drink (CHO+PRO, 60.5 g CHO + 14.5 g

PRO); and, placebo drink (PLA), offered during the cycling bout. Blood samples were

collected before, immediately after and 24 h after each test for creatine kinase (CK)

analysis. Isometric peak torque (PT) was measured before and 24 h after each condition.

The primary outcome was the time to complete the last 5km running section (t5km) in a

self-selected pace. Statistical differences were considered when p<0.05. Results: There

was no difference in t5km between CHO (1270.3 ± 130.5 s) vs. CHO+PRO (1267.2 ±

138.9 s) vs. PLA (1275.4 ± 120 s); p = 0.87; ES ≤ 0.1. Pre-post changes for PT and CK

values did not show differences in any of three conditions (p = 0.24, ES ≤ 0.4, p = 0.32,

0.3-1.04). Conclusion: For endurance sports lasting up to 2 h, with a pre-meal

containing 1.5 g/kg of CHO, CHO or CHO+PRO supplementation does not offer

additional benefits when compared to a PLA in performance and muscle recovery.

Keywords: Running. Cycling. Dietary Supplements. Sports Performance. Sports

Nutrition Sciences.

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INTRODUCTION

It has long been established that carbohydrate (CHO) ingestion during physical

exercise is effective in improving endurance performance due to its metabolic efficiency

for ATP resynthesis and the ability to reverse glycogen depletion during exercise

(Coggan and Coyle 1991; Jeukendrup et al. 2004). Current standard recommendations

from the American College of Sports Medicine (Thomas et al. 2016) suggest the

consumption of 30-60 g of CHO per hour, diluted in a 6-8% concentration, in events

lasting between 1 - 2.5 h, which has been supported by different studies (Medicine et al.

2000; Jeukendrup et al. 2011; Cermak and van Loon, 2013; Jeukendrup et al. 2014;

Thomas et al. 2016).

Recently, several studies have examined whether adding protein (PRO) to a

traditional CHO beverage in a ratio of CHO/PRO (4/1) would further enhance

endurance performance, with conflicting results have been reported (Ivy et al. 2003;

Williams et al. 2003; Saunders et al. 2004; Romano-Ely et al. 2006; van Essen and

Gibala 2006; Saunders 2007; Osterberg et al. 2008; Skillen et al. 2008; Valentine et al.

2008; Breen et al. 2010; Martínez-Lagunas et al. 2010; McGawley et al. 2012; Highton

et al. 2013). The argument that supports the hypothesis of an ergogenic effect with the

combination of CHO+PRO has been related to central fatigue attenuation and increased

protein synthesis, suggesting an attenuation of the exercise-induced muscle damage

(Spiller et al. 1987; Saunders et al. 2004). However, the physiological mechanism that

would explain the extra benefits can be considered contradictory, once carbohydrates

are the main energy sources at high-intensity exercise (van Loon et al. 1999; Gastin

2001; Osterberg et al. 2008). In this regard, the aforementioned position statement

(Thomas et al. 2016) does not mention the co-ingestion of CHO+PRO as a reliable

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strategy to improve endurance performance, whereas discusses its possible advantages

in muscle recovery. However, it has been shown that time-to-exhaustion (TTE) could be

improved by 13-36% under CHO+PRO supplementation (Jeukendrup et al. 1996) and

for muscle recovery, some investigators have reported an attenuation of creatine kinase

(CK) increased levels (Saunders et al. 2004; Millard-Stafford et al. 2005; Romano-Ely

et al. 2006; Saunders 2007; Skillen et al. 2008; Valentine et al. 2008; Gilson et al.

2010). Methodological differences in the studies, such as the use of TTE protocols,

which have a high coefficient of intra-test variation, and also the use of non-isocaloric

CHO and CHO+PRO supplementation make it difficult to discern whether the observed

benefits are a result of PRO per se or other factors (Stearns et al. 2010). Nevertheless,

evidence regarding CHO+PRO beverages consumption during exercise is still lacking

for similar conditions to that the athletes experience on the race day (i.e., a self-selected

pace protocol and adequate nutritional status especially).

Therefore, in order to investigate the contradictory findings in the literature

regarding the CHO+PRO supplementation during endurance exercise and to provide

evidence for the real-world setting, the present study aims to compare the effect of three

different nutritional supplementation strategies [CHO vs. CHO+PRO vs. Placebo

(PLA)] on performance and on muscle recovery. The primary outcome chosen was the

time to complete the last 5km running section (t5km) in a self-selected pace after

previously running (10 km) and cycling (40 km). Our hypothesis was that CHO+PRO

supplementation would be more effective in improving endurance performance than

CHO supplementation, and would also promote an attenuation of muscle damage.

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MATERIALS AND METHODS

Participants

Fifteen male amateur athletes volunteered to participate in the study. Two

participants dropped out for reasons unrelated to the study (one participant had an injury

not related to the study protocol and one participant moved to another country),

resulting in a final sample of 13 participants. Inclusion criteria were: age 18-45 years, a

minimal training routine of 10 h/week and previous participation in at least two sprints

or Olympic-distance duathlon/triathlon. We adopted as exclusion criteria the use of anti-

inflammatory drugs and the presence of previous serious skeletal muscle injuries. The

participants’ average weekly training volume was ~38 km of running, ~208 km of

cycling, ~9 km swimming and ~2 h of complementary training (i.e., core training,

strength training, etc.). Informed consent was obtained from all participants and all

procedures involved in the present study were approved by the University Institutional

Review Board (permit number 1.320.276), and conducted in accordance with the

Declaration of Helsinki. Physiological and anthropometric participants’ characteristics

are present in Table 1.

<<< Table 1 here >>>

Experimental Design

In a randomized, crossover, double-blind, placebo-controlled clinical trial,

participants were recruited from January to May 2016, through visits to local triathlon

clubs and invitations to athletes and coaches, presenting all the eligibility criteria.

Recruitment data are reported in Figure 1.

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<<< Figure 1 here >>>

Participants attended the Exercise Research Laboratory at the Federal University

of Rio Grande do Sul for a total of seven visits: one day of preliminary tests, three days

of simulated duathlon trials (SDT) where the supplementation was randomly

administrated (CHO, CHO+PRO or PLA), and three days of post-24h assessments

(POST24H). Also in the first visit the athletes were instructed on how to fill out a diet

record form.

Preliminary Tests

At the first visit, body mass and height (digital coupled stadiometer scale, OS-

180, Urano, Brazil) and skinfolds (Cescorf, Brazil) were measured and preliminary tests

of maximal isometric torque, incremental cycling and incremental running tests were

performed soon thereafter.

Maximum Isometric Torque Test

After warm-up and familiarization, the maximal isometric torque for the knee

extensors muscles of dominant limb was assessed on the isokinetic dynamometer

(Cybex Norm, Ronkonkoma, USA). Three attempts of 5 s were performed at a fixed

angle of 60° (0° = full extension knee). Recovery period between attempts was defined

in 120 s. The isometric peak torque value (PT) was considered as the highest value

reached during the three attempts, as determined by the equipment software.

Incremental Cycling Test

Fifteen minutes after PT measurements, an incremental cycling test (Lode

Excalibur Sport, Groningen, Netherlands) was performed to evaluate the maximal

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oxygen uptake (VO2max) and maximal power output (POmax). After cycle ergometer

components were adjust to the participants' body dimensions, a brief familiarization

with the equipment was conducted. Oxygen consumption was determined by a breath-

by-breath open circuit spirometry system (Quark CPET, Cosmed, Italy). The protocol

consisted of a 10-min warm-up at 100 W, followed by increments of 5 W every 15 s

until exhaustion. Participants were instructed to maintain an average cadence between

90-100 rpm and were verbally stimulated to perform maximum effort. Heart rate (HR)

was continuously measured (S610, Polar Electro Oy, Finland). The tests lasted 11.3 ±

1.4 min and were terminated in the inability to maintain the cadence above 70 rpm or by

participants’ volitional interruption. The test results were used to tailor the cycling

section workload during all the SDTs.

Incremental Running Test

An incremental running test was conducted on a treadmill (Quinton Instruments,

Seattle, USA) 30 min after the cycling test, in order to determine the intensity of the

first running section of the SDTs. The initial speed was 8 km/h and increments of 1

km/h every minute were performed. The HR was also continuously registered; tests

lasted 11.7 ± 0.9 min and were ended whenever participants met two or more of the

following criteria: volitional fatigue; a respiratory exchange ratio ≥ 1.15; HR ≥ 95% of

age-predicted maximum HR; or a plateau in oxygen consumption with increasing load.

VO2max, used only for characterization of the sample, was obtained through a visual

inspection of the graphs and ventilatory thresholds were determined by three

independent observers, as previously described (Cunha et al. 2016). On that same day,

the participants received a diet record form and were instructed to record all 24 h food

intake prior to the first SDT.

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Supplementation Regimens

Supplements administered included: (1) CHO: 75g maltodextrin; (2) CHO+PRO:

a solution of 75g (60.5g CHO + 14.5g PRO); (3) PLA: flavored placebo drink (0 kcal).

All supplements were diluted in cold water on the day of the trial. Details of

supplementation are given in Table 2.


All the supplements contained 450 mL, divided into three doses of 150 mL. They

were administered in non-see-through bottles at km 5, 20 and 35 of the cycling section.

A different flavor was used in each supplement in order to minimize the risk of taste

comparisons between beverages. Breen et al. (2010) showed that when the flavor is the

same, the participants are able to more easily identify the composition of the drink

(Breen et al. 2010). The CHO and CHO+PRO beverages had a concentration of 16.6%

and 13.4%, respectively. Although the classic recommendation is a 6-8% dilution, a

previous study (McGawley et al. 2012) utilized the concentration of 14.4% and had no

reports of gastrointestinal discomfort. The randomization of the supplementation order

was performed on a specialized website (randomization.com) by an independent

researcher not involved in the data collection and analyses.

Simulated Olympic-Distance Duathlons Trials

Three simulated Olympic-distance duathlon trials (SDT) were performed. The

participants arrived at the laboratory after an 8 h overnight fast and blood samples were

collected immediately. Soon after, the participants had breakfast and were asked by the

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dietitian about the diet and the training performed on the previous day. SDT comprised

10-km of treadmill running in constant-load intensity, 40-km of cycling on a cycle

ergometer (strategically arranged next to the treadmill) and 5-km of an outdoor running.

The last running section was performed outside to ensure a greater ecological validity.

Therefore, the first running section and the cycling section had controlled intensity and

were performed in the laboratory with controlled temperature (18 - 20ºC). The last

running section was treated as a self-selected pace time-trial, meaning that the time

spent by the participant to complete the 5 km (t5km) was the primary outcome

(performance) of the study. The trials were always performed at the same time of the

day to avoid circadian cycle variance and to ensure a standardized effect of the pre-

exercise meal; were performed on the same day of the week, to avoid variance of

previous and subsequent training intensity; and in similar climatic conditions. We asked

the participants to avoid strenuous training on the day before each trial.

Each SDT began with a 10-min warm-up on the treadmill on a self-selected pace.

The first running section of the SDT was performed at an intensity of 75% of the

maximum speed reached by the participant in the preliminary incremental running test.

Following the 10-km run, a period of up to 3-min transition was allowed before starting

the cycling section. For the 40-km cycling section, several intensity/time/distance ratios

were tested in previous pilot tests in order to find executable intensities that would

simulate the conditions during competitions. The section was therefore divided into

three periods of 13.33 km, each one with intensities at 60, 55 and 50% of the POmax

reached in the preliminary cycling test, respectively (Bernard et al. 2009).

Supplementation was administered during this 40-km cycling, as cycling has been

recognized as the best opportunity to eat during triathlon/duathlon events (McMurray et

al. 2006; Jeukendrup 2011). At the end of the cycling section, participants were given a

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further 3-min transition to prepare for the final run. Participants were verbally

stimulated to complete the 5-km run in the shortest possible time. Garmin devices

(Chicago, USA) were used by participants to delineate the distance and to record the

t5km. Participants received water ad libitum throughout the trial, although the total intake

was not controlled. Blood samples were collected immediately after each SDT.

Post-24 h Assessments

After 24 h (POST24H), participants returned to the laboratory for blood sample

collection and re-testing of knee extensor muscles’ PT, in similar procedures to those

previously described. During these 24 h, the participants were instructed not to perform

any kind of physical exercise and to match their previous days’ food consumption.

Participants were not fasted in the 24 h blood sample collection.

Diet Control

Participants were encouraged to maintain a standard diet during the period of the

study to avoid any nutritional bias. On the first visit, the participants received

instructions on how to fill out the diet record form. They were instructed to avoid eating

atypical foods and not to consume alcohol. On the first SDT, participants submitted

their diet record form to the dietitian (3-year practical experience, ~250 cases). The

participants were asked to match their food intake each day prior to a SDT and also over

the 24 h course after a SDT. Text messages were sent to remind them of what they had

eaten before. These data were later entered into an online software (AvaNutri, Brazil)

for nutritional analysis.

Pre-test Meal

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On each SDT day participants received a standard breakfast 45 minutes prior to

the trial start after an overnight fast. This standardization ensured an intake of the same

energy amount and proportion of macro and micronutrients in each one of the SDTs.

The meal was calculated following current recommendations for pre-exercise meals: 1.5

g/kg CHO. Each participant received two bananas, which weighed about 200 g and

contained ~50g of CHO. The remainder of the grammage (up to 1.5 g/kg) was given in

maltodextrin, diluted in water (10%).

Blood Samples and Analysis

Blood samples were collected from the antecubital vein into 4 mL

ethylenediamine tetraacetic acid (EDTA) tubes and centrifuged at 3500 rpm at 4°C for

10 min. The supernatant was then stored in a -80°C freezer for future analysis. CK

activity was measured using an automated analyzer (Cobas C111, Roche, Switzerland)

and commercially available kits (Roche Diagnostics, Switzerland). Blood samples were

obtained before, immediately after and 24 hours after each SDT in order to evaluate the

effect of the exercise protocol and the supplementations on the blood markers.

Sample Size Calculation

Sample size was planned to detect a moderate effect size (0.4 ES) on primary

outcome (t5km), considering a statistical power of 80% and type 1 error limit of 5% for a

repeated-measure design, resulting in a minimum of 12 subjects. A 15% addition was

set in order to support within-study participants’ drop-outs or exclusions, resulting in a

final sample size of 15 individuals.

Statistical Analysis

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Only the data from participants who completed all exercise interventions were

considered for analyses. The normality and sphericity of the data were evaluated by

Shapiro-Wilk and Mauchly tests, respectively. Greenhouse-Geisser Epsilon correction

factor was used for non-spherical data. Data were then presented as mean ± standard

deviation. A one-way ANOVA for repeated measures followed by a LSD post hoc test

was used to compare t5km between conditions (CHO vs. CHO+PRO vs. PLA). In

addition, a two-way ANOVA, followed by a LSD post hoc test, was used for the

comparison of the following variables (Peak torque = PRE and POST24H; CK = PRE,

POST, POST24H) between each one of the conditions (CHO vs. CHO+PRO vs. PLA).

The effect size (ES) was used for all variables, assuming values of 0.2; 0.5; > 0.8 for

low, moderate and high effects, respectively (Cohen, 1982). All analyses were

performed using the statistical package SPSS (Statistical Package for Social Sciences,

Chicago, USA) version 20.0, with a significance level of 5%.

RESULTS

Participants Flow

Invitation to participate of the present study was made to 50 athletes, out of

whom 46 responded to the recruitment call and 30 participated of the initial procedures.

Fifteen athletes were excluded for the following reasons: three did not meet inclusion

criteria, ten refused to participate and two got injured not related to the study. After this

stage, the remaining 15 athletes started the tests and 13 made through the whole process

and were included for the statistical analyses of both primary and secondary outcomes

(for further information, see the CONSORT flow diagram).

Nutritional Specifications

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The pre-SDT meal that was offered to all participants presented ~450 kcal, and

did not differ over the three conditions. The average meal’s nutritional parameters

calculated based on the participants’ average body mass (73.1 kg = 110g CHO) were:

200g of bananas (52g of CHO; 216 kcal); 58g of maltodextrin diluted in plain water at a

fixed concentration of 10% (58g of CHO; 232 kcal). The total amount of CHO and

energy intake were 110g and 448kcal, respectively. No gastrointestinal discomfort was

or any adverse event was experienced by the athletes.

The analyses of the food pattern of the participants, obtained from their diet

record form, revealed a daily energy intake of ~ 3100 kcal/day and a macronutrient

distribution of ~50.2% CHO (386.7 ± 105.2 g/day), 28.6% lipid (98.3 ± 34.8 g/day) and

21.3% PRO (168.7 ± 66.6 g/day). When asked about the order of supplementation they

had received throughout the study, only two of the thirteen participants (~ 15% of the

sample) were able to identify it correctly in a posteriori verification.

Performance

The external temperature did not show significant differences during the three

conditions: CHO (22 ± 7 ° C) vs. CHO+PRO (18 ± 7 ° C) vs. PLA (21 ± 6 ° C); P =

0.26; ES ≤ 0.5. Participants completed the total distances of the Olympic duathlon in ~

1h51min. Table 3 shows the general details of each SDT section.


The results of the present study showed that there were no differences (p = 0.87;

ES ≤ 0.1) in the final running performance (t5km) between CHO (1270.3 ± 130.5 s) vs.

CHO+PRO (1267.2 ± 138.9 s) vs. PLA (1275.4 ± 120 s) conditions. Figure 2 shows the

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mean ± SD of the individual performance values (t5km) in the different conditions.


Muscle Damage Markers

Isometric Peak Torque

Isometric PT did not change (p = 0.24) from pre vs. post SDT using each one of

three different nutritional strategies [PRE (302.2 ± 52.8 N.m) vs. POST24H CHO

(300.1 ± 41.4 N.m; ES = 0.04) vs. POST24H CHO+PRO (292.2 ± 49.4 N.m, ES = 0.19)

vs. POST24H PLA (282.1 ± 43.1 N.m, ES = 0.41)]; Figure 3.

<<< Figure 3 about here >>>

CK Concentrations

Increased CK levels immediately after the SDT for all nutritional conditions was

observed: [CHO (53%, p < 0.01, ES = 0.30); CHO+PRO (38%, p < 0.01, ES = 0.43)

and PLA (69%, p < 0.01, ES = 0.59)]. Similarly, in the PRE vs. POST24H, an increase

in CK concentrations was found for all conditions: [CHO (300%, p < 0.01, ES = 0.93);

CHO+PRO (82%, p < 0.01, ES = 0.73) and PLA (190%, p = 0.01, ES = 1.04)].

However, only the CHO condition showed an increased CK concentration between

immediately after (POST) vs. POST24H (139%, p = 0.02, ES = 0.67). No significant

differences were found between the different nutritional conditions (CHO vs.

CHO+PRO vs. PLA) in the different moments - pre, immediately after and after 24 h (p

= 0.32, ES ≤ 0.75); Figure 4.


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DISCUSSION

The main finding of the present study was that supplementation strategies using

CHO and CHO+PRO were not more effective than the placebo drink in the final’s race

performance (t5km). In addition, both supplements did not prevent the attenuation of the

exercise induced muscle damage better than placebo supplementation, since PT and

plasma CK concentrations showed no difference between conditions 24 hours after each

SDT. Thus, for endurance exercises of up to 2 h at ~80% of VO2max, the

supplementation of CHO and CHO+PRO are unable to show greater ergogenic and

muscle damage attenuation benefits than those demonstrated by the ingestion of a

placebo drink. These results were observed with SDT protocols preceded by a meal

containing 1.5 g/kg CHO, and with muscle glycogen stores potentially full, which

represents a real world setting for the race-day competition.

Although it is generally accepted that CHO intake during exercise is a good

ergogenic strategy (Coggan and Coyle 1991; Tsintzas et al. 1993; Coyle 2004;

Jeukendrup 2004; Jeukendrup 2011; McGawley et al. 2012; Cermak and van Loon

2013; Jeukendrup 2014; Thomas et al. 2016), previous studies have found mixed effects

regarding CHO+PRO supplementation and endurance performance enhancement

(Madsen et al. 1996; Ivy et al. 2003; Williams et al. 2003; Saunders et al. 2004;

Saunders 2007; Saunders et al. 2007; Osterberg et al. 2008; Valentine et al. 2008; Breen

et al. 2010; Stearns et al. 2010; Highton et al. 2013). In view of the higher incidence of

all types of gastrointestinal complaints and especially the high cost that these strategies

represent to athletes, it is questioned whether they are in fact necessary (McLellan et al.

2014). Potential positive effects of protein ingestion, such as glycogen sparing,

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reduction of muscle damage and central fatigue, have led researchers to hypothesize if

combining CHO+PRO during exercise would improve endurance performance.

However, in the present study, no ergogenic effect was observed when combining

CHO+PRO, and this result is in agreement with previous studies (Madsen et al. 1996;

Romano-Ely et al. 2006; van Essen and Gibala 2006; Osterberg et al. 2008; Valentine et

al. 2008; Breen et al. 2010; Martínez-Lagunas et al. 2010; McLellan et al. 2014; van

Loon 2014).

The present findings however, contrast with some recent studies (Ivy et al. 2003;

Saunders et al. 2004; Saunders 2007; Saunders et al. 2007) and their methodological

limitations may explain these discrepancies. There are two methods to evaluate

endurance performance: time-trial (TT) or time-to-exhaustion (TTE) protocols. TTE

protocols are the most commonly used protocols in current literature to evaluate

performance (Jeukendrup et al. 1996; Stearns et al. 2010). They are mainly useful when

searching for explanations for fatigue, but the observation that CHO or CHO+PRO

improves TTE does not necessarily mean that this strategy would also improve

performance in other exercise situations when the exhaustion is not necessarily reached.

TTE tests represent low ecological validity and have a high variation (about 26%)

between one test and another (Jeukendrup et al. 1996; Currell and Jeukendrup 2008).

TT protocols are more difficult to conduct and to control, but they are recognized as

being more environmental friendly and considered highly reproducible (Jeukendrup et

al. 1996; Currell and Jeukendrup 2008; McLellan et al. 2014). Another considerable

detail for the inconsistency between the findings of the present study and others is the

fact that most studies are not placebo-controlled (Stearns et al. 2010). Also, the lack of

isocaloric arms on trials is potentially a relevant methodological limitation (Stearns et

al. 2010; McLellan et al. 2014).

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In addition, the argument from previous studies that CHO supplementation

represents, under any conditions, a powerful ergogenic effect must be rethought

(Thomas et al. 2016). The conditions prior to exercise were well controlled in the

present study and our findings showed that current CHO recommendations should be

questioned, at least for events up to 2 h when subjects are nourished. In fact, exercise

duration and pre-exercise nutritional status seems to be a determining factor in this

discussion.

Current studies are usually performed with duration longer than 2 h, where there

seems to be a consensus that supplementation is important (Cermak and van Loon

2013). Indeed, few studies investigated the actual effect of supplementation on

relatively shorter activities such as Olympic-distance events. To our knowledge, only

two studies were conducted investigating Olympic triathlon, and none had simulated an

Olympic duathlon before (Millard-Stafford et al. 1990; McGawley et al. 2012).

McGawley et al. (2012) in a simulated trial of Olympic triathlon with protocols very

similar to ours - first and second section of the test with controlled intensities and last

section treated as a TT - reported a final time 4.0 ± 1.3% lower in the CHO condition

compared to PLA (p = 0.010). However, Millard-Stafford et al. (1990) did not observe

significant differences in the performance of triathletes submitted to a simulated

triathlon protocol and consuming placebo or 7% CHO solution. For these authors, once

pre-exercise muscle glycogen levels are elevated, the demand for exogenous CHO

sources is sufficiently addressed by muscle glycogen stores, and additional increases in

blood glucose may not influence the performance (Millard-Stafford et al. 1990). In this

regard, Flynn, Costill et al. (1987), in a 2 h cycling protocol, raised the hypothesis of the

"not so long" exercise duration, where the authors reported absence of CHO’s ergogenic

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effect when subjects have plenty of glycogen stored (500g CHO/day for the previous 48

h before exercise, pre-race wet muscle glycogen of ~ 175 mmol/kg), which may also be

valid for the finding of the present study (Flynn et al. 1987). It is possible to speculate

that the pre-exercise meal might have played a role for the lack of differences between

CHO supplementation and PLA. It was administrated higher CHO concentrations (1.5

g/kg) than the minimum recommended by the current guidelines (Thomas et al. 2016)

recommendation (1 g/kg), therefore it is possible that muscle glycogen stores before

exercise were potentially fulfilled, although not measured in the present study. None of

these studies (Flynn et al. 1987; Millard-Stafford et al. 1990; McGawley et al. 2012)

compared the effect of the CHO+PRO combination.

Current guidelines (Jeukendrup 2011; Jeukendrup 2014; Thomas et al. 2016)

strongly recommend CHO intake during exercise, but many do not take into

consideration what the athlete usually consumes. Some authors have already observed

that during real-life competitions, athletes do not consume the volume of fluid typically

used in experimentally controlled studies (Millard-Stafford et al. 1990; Burke et al.

2005). It seems to happen due to factors involving nutritional beliefs and personal

experiences. Some athletes prefer not to ingest large fluid quantities because they know

they will have to slow down to obtain and consume the beverage. A survey with the

participants was performed in the present study and the average intake of CHO drinks in

a race was ~475mL. Millard-Stafford et al. (1990) also performed a survey and found

similar total volume (400 mL).

Regarding the indirect markers of muscle damage, the present results showed

that the isometric PT did not differ from the PRE (basal) to vs. POST24H for any

nutritional condition. Similarly, the supplementation of CHO vs. CHO+PRO vs. PLA

also did not produce different effects in CK 24 h after the SDT. Although the results of

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plasma CK levels changed at different moments (PRE vs. POST vs. POST24H).

However, the isometric PT was not significantly changed, indicating that some level of

muscle damage was induced by the exercise protocols but was not significant from the

functional impairment perspective. The participants of the present study were extremely

trained (despite not been professional athletes) and were used to perform cycling and

running activities, therefore it is reasonable to assume that their training status conferred

them protection against muscle damage (Newton et al. 2008). However, despite no

differences found in isometric PT from PRE to POST24H, alterations were found in CK

levels, indicating that some muscle damage to the muscle structures have occurred.

Nevertheless, the results found in the present study regarding CK response must the

treated with caution, since CK is a blood marker that has high variability and it has

already been reported dissociated time course of changes between isometric strength

and CK responses (Nosaka et al. 2006). We cannot guarantee that there is no effect of

only protein intake on muscle recovery, and observing that significant muscle damage

was not found in the present study, the protein intake on muscle recovery could not be

fully tested. However, our results suggest that the ingestion of a mixed drink

(CHO+PRO) during exercise does not accelerate muscle recovery process, once CK

values were increased 24 h post-exercise. Additionally, it has been reported that the

intake of protein throughout the day seems to be much more important than

supplementation in a single moment (van Loon 2014).

One limitation of the present study was the impossibility to familiarize the

subjects with the protocol. Only two of the thirteen participants had the first SDT as

their best test, demonstrating a possible learning effect throughout the study. Another

limitation was the physical condition that each participant arrived at each test day.

Besides the fatigue condition, their competition calendar also prevented the STDs from

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being performed within a one-month interval. It is believed, however, that these are all

common limitations when studying this type of population. Nonetheless, future studies

should evaluate muscle glycogen content by needle biopsy to better understand the

underpinned physiological aspects related to the supplementation regimens. To the best

of our knowledge, the present study was the first to compare the effects of CHO vs.

CHO+PRO vs. PLA supplementation on performance and muscle damage rates of

endurance athletes through a real-world experimental model. A duathlon race protocol

was chosen, since the run-cycling-run pattern represents a pronounced intense muscular

activity and subsequently muscle damage. We truly believe that our findings could be

extrapolated to any endurance activity of up to 2 h.

Conclusion

For endurance sports of up to 2 h in duration preceded by a meal containing 1.5

g/kg of CHO, the use of CHO and CHO+PRO supplements do not offer extra benefits

when compared to a placebo drink with respect to endurance performance and muscle

recovery of amateur athletes. Given that supplementation implies high costs to the

athletes, this strategy could be considered futile in this scenario, representing an

unnecessary practice for a real-world setting.

The authors have no conflicts of interest to report.

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Table 1. Participants’ physiological and anthropometric characteristics (n = 13), expressed as mean ± standard deviation (SD).

Variable Mean ± SD

Age (yr) 29.7 ± 7.7

Body mass (kg) 73.1 ± 7.5

BMI (kg/m2) 23.1 ± 1.4

Fat (%) 7.9 ± 2.5

VO2max on treadmill (mL/kg/min) 62.2 ± 5.4

Vmax on treadmill (km/h) 19.7 ± 0.9

VO2max on cycle ergometer (mL/kg/min) 61.9 ± 5.2

POmax (W) 376.5 ± 28

BMI = Body Mass Index; VO2max = maximal oxygen uptake; POmax = maximal power output, obtained in incremental cycling test; Vmax = maximal speed, obtained in incremental running test. Skinfolds used to calculate body fat percentage: triceps, subscapular, axillary, pectoral, suprailiac, abdominal, thigh (7-fold protocol of Pollock et al. 1978).

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Table 2. Nutritional information for the supplementation.

Product CHO

Body Action

Maltodextrin

CHO+PRO

CarbPro 4:1 Recovery, Essential Nutrition

PLA

Clight

Flavor Acai Lemon Orange

Dose (g) 75 75 7

Energy (kcal) 300 300 0

Carbohydrate (g) 75 60.5 0

Protein (g) 0 14.5 0

Sodium (mg) 0 26.2 36

CHO = carbohydrate supplementation only; CHO+PRO = carbohydrate + protein combined supplementation; PLA = placebo supplementation.

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Table 3. General details of each SDT section (n=13), expressed as mean ± standard deviation (SD).

Section Characteristics

First running Speed (km/h) 10-km running 14.8 ± 0.7

Total time (min) 10-km running 40.8 ± 2.1

PLA: time (s) 10-km running 2449 ± 124

CHO: time (s) 10-km running 2447 ± 113

CHO+PRO: time (s) 10-km running 2457 ± 111

T1 (min) 2.5 ± 0.5

Cycling PO (W) 0 – 13.3 km 225.9 ± 16.8

PO (W) 13.4 – 26.6 km 207.3 ± 15.5

PO (W) 26.7 – 40 km 188.4 ± 14.1

Total time (min) 40-km cycling 49.1 ± 4.2

PLA: time (s) 40-km cycling 2931 ± 261

CHO: time (s) 40-km cycling 2944 ± 262

CHO+PRO: time (s) 40-km cycling 2943 ± 260

T2 (min) 2.2 ± 0.8

Second running t5km (min) 21.2 ± 2.1

T1 = First transition: running section to cycling section; T2 = Second transition: cycling section to second running section; PO = Power output; t5km = time to complete the 5-km run (last running section); and comparison of time first running (10-km) and cycling (40-km), with consumption of PLA = placebo supplementation; or CHO = carbohydrate supplementation only; or CHO+PRO = carbohydrate + protein combined supplementation (p > 0.05).

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Figure captions

Figure 1: The flow of the participants through the trial.

Figure 2. Performance of the last running section (t5km) for all trial conditions. (p >

0.05). Data are expressed in seconds and as mean ± SD.

Figure 3. Values of isometric peak torque (PT) from basal conditions (PRE) vs.

POST24H for all trial conditions. (p > 0.05). Data are expressed in N.m. and as mean ±

SD.

Figure 4. PRE, POST and POST24H differences for serum creatine kinase (CK) levels.

* = difference to the PRE moment (p ≤ 0.01); † = difference to the immediately-after

moment (POST) (p = 0.02). Data are expressed as U/L and as mean ± SD.

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Figure 1: The flow of the participants through the trial.

102x80mm (300 x 300 DPI)

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Figure 2. Performance of the last running section (t5km) for all trial conditions. (p > 0.05). Data are expressed in seconds and as mean ± SD.

147x82mm (300 x 300 DPI)

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Figure 3. Values of isometric peak torque (PT) from basal conditions (PRE) vs. POST24H for all trial conditions. (p > 0.05). Data are expressed in N.m. and as mean ± SD.

176x107mm (300 x 300 DPI)

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Figure 4. PRE, POST and POST24H differences for serum creatine kinase (CK) levels. * = difference to the PRE moment (p ≤ 0.01); † = difference to the immediately-after moment (POST) (p = 0.02). Data are

expressed as U/L and as mean ± SD.

146x81mm (300 x 300 DPI)

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Draft - University of Toronto T-Space€¦ · Draft 2 ABSTRACT Introduction: Protein (PRO) combined with a carbohydrate (CHO) beverage may have an ergogenic effect on endurance performance