Eur J Appl Physiol
DOI 10.1007/s00421-011-1831-5
ORIGINAL ARTICLE
Combined carbohydrate–protein supplementation improves
competitive endurance exercise performance in the heat
Andrew J. Cathcart • Scott R. Murgatroyd •
Alison McNab • Laura J. Whyte • Chris Easton
Received: 7 May 2010 / Accepted: 7 January 2011
Ó Springer-Verlag 2011
Abstract Laboratory-based studies have demonstrated
that adding protein (PRO) to a carbohydrate (CHO) sup-
plement can improve thermoregulatory capacity, exercise
performance and recovery. However, no study has inves-
tigated these effects in a competitive sporting context. This
study assessed the effects of combined CHO–PRO sup-
plementation on physiological responses and exercise
performance during 8 days of strenuous competition in a
hot environment. Twenty-eight cyclists participating in the
TransAlp mountain bike race were randomly assigned to
fitness-matched placebo (PLA 76 g L-1 CHO) or CHO–
PRO (18 g L-1 PRO, 72 g L-1 CHO) groups. Participants
were given enough supplements to allow ad libitum con-
sumption. Physiological and anthropometric variables were
recorded pre- and post-exercise. Body mass decreased
Communicated by Jacques R. Poortmans.
This paper is dedicated to the memory of Dr Andy Cathcart, who was
tragically killed in a cycling accident prior to the submission of this
manuscript. He will be sorely missed by his colleagues, the students
he taught, and his family and friends.
A. J. Cathcart
A. McNab
L. J. Whyte
Institute for Diet, Exercise and Lifestyle,
Faculty of Biomedical and Life Sciences, University of Glasgow,
Glasgow G12 8QQ, UK
S. R. Murgatroyd
Institute of Membrane and Systems Biology,
Faculty of Biological Sciences, University of Leeds,
Leeds LS2 9JT, UK
C. Easton (&)
School of Life Sciences, Faculty of Science,
Kingston University, Penryhn Road,
Kingston upon Thames KT1-2EE, UK
e-mail: C.Easton@Kingston.ac.uk
significantly from race stage 1 to 8 in the PLA group
(-0.75 ± 0.22 kg, P = 0.01) but did not change in the
CHO–PRO group (0.42 ± 0.42 kg, P = 0.35). Creatine
kinase concentration and muscle soreness were substan-
tially elevated during the race, but were not different
between groups (P = 0.82, P = 0.44, respectively). Urine
osmolality was significantly higher in the CHO–PRO ver-
sus the PLA group (P = 0.04) and the rise in tympanic
temperature from pre- to post-exercise was significantly
less in CHO–PRO versus PLA (P = 0.01). The CHO–PRO
group also completed the 8 stages significantly quicker
than the PLA group (2,277 ± 127 vs. 2,592 ± 68 min,
respectively, P = 0.02). CHO–PRO supplementation
therefore appears to prevent body mass loss, enhance
thermoregulatory capacity and improve competitive exer-
cise performance despite no effect on muscle damage.
Keywords Cycling
Recovery
Sports drinks
Fatigue

Endurance
Introduction
The efficacy of exogenous carbohydrate (CHO) supple-
mentation to improve endurance performance during
events in relatively thermo-neutral environments lasting
from 1 h to many days has been well documented (Coyle
et al. 1986; Febbraio et al. 2000; Fielding et al. 1985;
Jeukendrup et al. 1997; Mitchell et al. 1989; Tsintzas et al.
1996). However, the facility of other macronutrients, sup-
plemented either alone or with CHO, to improve perfor-
mance is still widely debated (Pitsiladis et al. 1999;
Romano-Ely et al. 2006; Saunders et al. 2004; Whitley
et al. 1998). Despite, or perhaps because of this, there has
been a recent surge of interest in the possible ergogenic
123

benefit of adding small quantities of protein (PRO) to CHO
supplements during exercise (Berardi et al. 2008; Saunders
et al. 2007, 2009; Toone and Betts 2010).
This line of research likely stems from several publi-
cations reporting enhanced glycogen resynthesis post-
exercise and improved performance during subsequent
exercise when PRO was consumed with CHO (CHO–PRO)
immediately post-exercise (Ivy et al. 2002; Williams et al.
2003). However, the mode of action for this benefit is
thought to be an amino acid stimulated augmented insulin
release (Reed et al. 1989; Zawadzki et al. 1992); a mech-
anism that is unlikely to offer any additional physiological
advantage (over CHO ingestion alone) during the exercise
per se. Despite this, several authors have in fact demon-
strated improvements in time to exhaustion at both fixed
(Saunders et al. 2004, 2007) and variable (Ivy et al. 2003)
exercise work rates and late-exercise time-trial perfor-
mance (Saunders et al. 2009) in subjects consuming CHO–
PRO compared to CHO-only supplements (matched for
CHO not calorie content). It should be noted, however, that
not all studies have been able to demonstrate a benefit of
CHO–PRO supplementation over that of CHO-only when
matched for either CHO (van Essen and Gibala 2006) or
calorie content (Romano-Ely et al. 2006). Indeed, Toone
and Betts (2010) recently reported that supplementation
with CHO–PRO significantly slowed cycling time-trial
performance compared with isocaloric CHO.
It remains unclear, therefore, as to whether adding cal-
orie content by the addition of PRO or replacing a portion
of the calorie content of CHO with PRO has any ergogenic
benefit on single bouts of endurance exercise. However, the
consumption of PRO in combination with CHO post-
exercise has been shown to enhance post-exercise muscle
glycogen resynthesis (Ivy et al. 2002; Williams et al.
2003), reduce muscle damage (Cockburn et al. 2008;
Saunders et al. 2004) and increase plasma volume and
thermoregulatory capacity (Goto et al. 2007; Ozaki et al.
2009). This last point is particularly intriguing as some
(Davis et al. 1988; Febbraio et al. 1996; Millard-Stafford
et al. 1990) though not all (Below et al. 1995; Carter et al.
2003; Millard-Stafford et al. 1992) studies have demon-
strated the benefits of CHO supplementation during exer-
cise to become less marked, or even disappear, compared
to plain water in hot environments. Therefore, it is plau-
sible that the addition of PRO to CHO supplements may
only become ergogenic when the exercise undertaken is
sufficient to challenge the body’s ability to resynthesise
muscle glycogen, to repair exercise-induced muscle dam-
age and/or to thermoregulate. To our knowledge no study
has yet to examine such effects during competitive exercise
performance. Thus, the current study aimed to investigate
whether the addition of PRO to a CHO supplement during
an intense, prolonged, multi-day cycling competition in a
123
Eur J Appl Physiol
hot environment would enhance thermoregulation, reduce
muscle damage and, ultimately, improve performance.
Methods
Subjects
Twenty-eight trained mountain bikers (Jeukendrup et al.
2000) (4 female and 24 male, Table 1) volunteered and
provided written informed consent to participate in the
study (approved by the Faculty of Biomedical and Life
Sciences, University of Glasgow Ethics Committee). All
procedures were conducted in accordance with the Decla-
ration of Helsinki. Each of the recruited participants was a
competitor in the 2007 TransAlp Challenge mountain bike
race. This competition is an annual multi-day event during
which participants are required to cross the Central Alps in
eight consecutive days/stages, with each stage consisting of
one cross-country marathon (Table 2). It is for this reason
that the TransAlp Challenge is considered to be one of the
most difficult cross-country marathon races in the world
(Wirnitzer and Kornexl 2008). Ambient temperature and
relative humidity at the end of each race stage averaged
33°C (range 29–40°C) and 42% (range 29–51%), respec-
tively. Five subjects withdrew from the race before com-
pletion and were therefore excluded from the study.
Experimental procedures
Day 0
On the day prior to the race commencing, subjects reported
to the field lab and performed a three-stage progressive
submaximal test for prediction of peak oxygen uptake
_ 2 peak ) (Åstrand and Ryhming 1954) on a computer-
(VO
controlled electromagnetically braked ergometer that uti-
lised the subjects own bicycle (Cyclus II, H/P Cosmos,
Nussdorf, Germany). The subjects were then randomly
assigned to one of two fitness-matched groups, placebo
(PLA) or CHO–PRO, on the basis of predicted VO _ 2 peak .
Table 1 The physical characteristics of the two groups
Placebo group CHO–PRO group
(n = 10) (n = 13)
Age (years) 31 ± 1 32 ± 1
Height (cm) 181 ± 2 177 ± 2
Pre-race body mass (kg) 76.9 ± 2.6 72.6 ± 2.3
_ 2 peak
Predicted VO 63.7 ± 2.6 64.0 ± 2.4
(ml kg-1 min-1)
Data presented as the mean ± SEM
Eur J Appl Physiol

Table 2 Distance and total

Stage location Distance (km) Elevation (m) Temperature (°C) Humidity (%)
elevation of vertical ascents on
each stage and ambient Stage 1: Füssen-Imstl 80.1 1,962 34 38
temperature and humidity at the
end of each stage Stage 2: Imstl-Ischgl 76.4 3,171 40 29
Stage 3: Ischgl-Scoul 75.3 2,547 35 38
Stage 4: Scoul-Livigno 77.2 2,621 31 41
Stage 5: Livigno-Naturns 122.2 2,909 28 48
Stage 6: Naturns-Kaltern 97.4 3,930 29 51
Stage 7: Kaltern-Andalo 74.6 3,071 30 47
Average temperature and Stage 8: Andalo-Riva 62.2 1,480 34 42
humidity are presented as the Total/average 665.4 21,691 33 ± 1 42 ± 3
mean ± SEM

The four female subjects were divided equally between
PLA and CHO–PRO groups.
Days 1–8 (pre-exercise)
Each morning of the race subjects reported to the field lab
between 6 and 9 a.m. following an overnight fast where
they were weighed using digital scales (Model 888, Seca,
Hanover, MD, USA), reported palpated muscle soreness on a
10-point visual analogue scale (0, no soreness; 10, extreme
soreness; Berardi et al. 2008) and provided a urine sample. A
heart rate (HR) monitor (S610i, Polar Sports, Kempele,
Finland) was attached to record HR continuously throughout
each stage and indicate peak HR (HRpeak). Tympanic
membrane temperature (Tty) was recorded using a tympanic
membrane thermometer (First Temp Genius Thermometer,
Sherwood-Davis and Geck, St Louis, MO, USA) [coefficient
of variation (CV) \3%]. The same investigator performed
each Tty measurement by pulling the ear upwards and
backwards while inserting the probe as far as possible into
the ear until a tight fit was achieved (Easton et al. 2007). Tty
was recorded in rectal equivalent mode that utilises an
algorithm to predict rectal temperature. The manufacturers
calibrated the thermometer and the calibration was validated
by immersion in a water bath at three temperatures: 30, 35
and 40°C. Additionally on days 1 and 8 subjects provided a
small (\100 lL) fingertip capillary blood sample.
Days 1–8 (post-exercise)
Immediately after completing each stage, subjects reported to
the field lab where they were re-weighed, reported palpated
muscle soreness, provided a urine sample and Tty was recor-
ded. Additionally on days 1, 3, 5, 7 and 8 they also provided a
small (\100 lL) fingertip capillary blood sample.
Blood and urine analysis
Capillary blood samples were analysed immediately on
collection using automated analysers for concentrations of
glucose ([glucose]: days 1 and 8, pre- and post-exercise)
(Accu-chek, Roche, Sussex, UK) (CV \3%) and creatine
kinase ([CK]: day 1 pre- and post-exercise, and days 3, 5, 7
and 8 post-exercise) (Reflotron, Biostat, Stockport, UK)
(CV \4%). The same investigator performed the analysis
on each occasion and analyser performance was checked
daily according to the manufacturer’s instructions against
the supplied calibration standards. Urine samples were
stored between 0 and 5°C and subsequently analysed in
duplicate within 12 h for osmolality (Micro-osmometer
3300, Vitech Scientific, Sussex, UK) (CV \2%).
Supplementation
The participants in both groups were supplied with suffi-
cient solid and liquid supplements on the morning of each
stage to permit ad libitum consumption during every stage
based on the anticipated duration. For the PLA group liquid
supplements contained 76 g CHO, 460 mg Na? and
120 mg K?: all L-1 while solid supplements contained
37 g CHO, 3 g whey PRO, 5 g fat and 55 mg Na?:
all bar-1. For the CHO–PRO group liquid supplements
contained 72 g CHO, 18 g whey PRO, 648 mg Na? and
170 mg K?: all L-1 while solid supplements contained
24 g CHO, 13 g whey PRO, 4 g fat and 100 mg Na?:
all bar-1. All commercially available supplements were
flavour-matched (choice of berry or citrus) and provided in
generic packaging, marked only with a ‘1’ or ‘2’, so that
both subjects and researchers were unaware of the sup-
plement contents. Participants were free to consume as
much solid and liquid supplements as they required during
each stage but were asked to refrain from consuming
anything other than water and highly water based fruit (e.g.
watermelon) from the on-course feed stations. Addition-
ally, they were asked to abstain from alcohol consumption
and any other nutritional supplementation throughout the
period of the study. Outwith the duration of each stage
competitors’ breakfast and evening meals were provided
by the race organisers. The subjects in both groups were
instructed to eat as much as they required from the meals

123

provided and were asked to record their entire dietary
intake via a daily 24 h recall diary. These diaries were
analysed using computerised dietary analysis software
(CompEat Pro V5.8.0, Lifeline Nutritional Services Ltd,
London, UK) which provided information on daily energy
intake and macro-nutrient content. Only 12 of the partici-
pants (6 in the PLA group and 6 in the CHO–PRO group)
returned completed diaries and were included in the dietary
analysis.
Statistical analysis
Data are reported as mean and standard error (mean ± -
SEM). Differences in physical characteristics, dietary
analysis and race completion time between supplementa-
tion groups were assessed using independent t tests. All
remaining effect data were analysed using a two-way
[1 between (group) and 1 within (race stage)] or three-way
[1 between (group) and 2 within (race stage and time)]
ANOVA with repeated-measures to examine the effects of
group (PLA vs. PRO), race stage (1–8) and time (pre- or
post-exercise). Subsequent post hoc tests to determine
significant differences in various pairwise comparisons
were performed using the Bonferroni test. The null
hypothesis was rejected when P \ 0.05. 95% confidence
intervals (95% CI) are included together with P values,
where appropriate. All statistical procedures were com-
pleted using SPSS for Windows version 17.0.
Results
Physical characteristics
There were no significant differences in age (P = 0.82),
height (P = 0.18), pre-race body mass (P = 0.24) or
Table 3 Mean daily
supplementation and dietary
analysis of the two groups During the race
Liquid supplement ingestion rate (L h-1)
Solid supplement ingestion rate (g h-1)
-1
Caloric intake (kcal h )
Carbohydrate (CHO) intake (g h-1)
Protein (PRO) intake (g h-1)
Fat intake (g h-1)
Pre- and post-race
-1
Caloric intake (kcal day )
Carbohydrate (CHO) intake (g day-1)
Data presented as mean ± SEM Protein (PRO) intake (g day-1)
* Significant difference Fat intake (g day-1)
(P \ 0.05) between groups
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Eur J Appl Physiol
_ 2 peak (P = 0.95) between PLA and CHO–
predicted VO
PRO supplementation groups (Table 1).
Supplementation and dietary analysis
There were no significant differences in the mean rate at
which liquid (P = 0.11) and solid (P = 0.14) supplements
were ingested during the race (Table 3). The mean rates of
CHO and fat intake during the race were not significantly
different between the two groups (P = 0.24 and P = 0.16,
respectively) (Table 3). Subjects in the CHO–PRO group
ingested PRO at a significantly greater rate than in the PLA
group (P \ 0.01, 95% CI 11.94–18.13 g h-1) and had a
significantly higher energy intake rate (P = 0.03, 95% CI
7.77–131.96 kcal h-1) (Table 3). Outwith the race, there
were no significant differences in mean daily caloric
(P = 0.82), CHO (P = 0.34), PRO (P = 0.52) or fat
(P = 0.23) intakes between the two groups of subjects
(n = 6 in both groups).
Body mass
A repeated-measures ANOVA revealed a significant main
effect for time (P \ 0.01) and group 9 race stage inter-
action (P = 0.04) with no significant differences between
supplementation groups (P = 0.33). There was a signifi-
cant decline in body mass from pre- to post-exercise on all
race stages (all P \ 0.01) with no significant differences
between groups. The mean change in body mass from pre-
to post-exercise for all stages (kg and % of pre-exercise
mass) were -1.3 ± 0.1 kg and -1.8 ± 0.2% for PLA, and
-1.4 ± 0.1 kg and -2.0 ± 0.2% for CHO–PRO. In the
PLA group there was a significant decrease in both pre-
exercise (-0.75 ± 0.22 kg, P = 0.01, 95% CI -0.25 to
-1.25 kg) and post-exercise (-0.69 ± 0.26 kg, P = 0.03,
95% CI -0.11 to -1.27 kg) body mass from race stage
1–8 whereas a small, but non-significant rise in body mass
Placebo group CHO–PRO group
(n = 10) (n = 13)
0.53 ± 0.05 0.69 ± 0.08
20.31 ± 2.54 14.00 ± 3.02
230 ± 10* 300 ± 27*
52.52 ± 2.64 56.40 ± 4.98
1.02 ± 0.13* 16.06 ± 1.39*
1.70 ± 0.21 1.18 ± 0.32
(n = 6) (n = 6)
2,831 ± 157 2,772 ± 207
350.1 ± 18.9 386.5 ± 30.8
118.7 ± 8.8 110.5 ± 8.6
113.4 ± 9.4 95.7 ± 10.3
Eur J Appl Physiol

Pre-Exercise Post-Exercise 1400

Day 1 Day 8 †
82
1200
PLA
80 *
† CHO-PRO

Creatine Kinase (Ul⋅L-1)

* 1000
†
Mean Body Mass (kg)

78

800 †
76
† †
74
600 †
†
72
400
70

200
68

66 0
PLA CHO-PRO PLA CHO-PRO 1 1 3 5 7 8
(pre) (post) (post) (post) (post) (post)

Fig. 1 Body mass pre- and post-exercise on race stages 1 and 8 in the
PLA and CHO–PRO groups. Data are presented as the mean
(columns) ± SEM (error bars); *significant difference from stage 1;
significant difference from pre-exercise
was observed in the CHO–PRO group (pre-exercise
0.42 ± 0.42 kg, P = 0.35; post-exercise 0.31 ± 0.36 kg,
P = 0.42) (Fig. 1).
Muscle damage
Both groups commenced the race with similar circulating
levels of CK within the normal physiological range (PLA
130.6 ± 26.1 U L-1 vs. CHO–PRO 130.9 ± 22.9 U L-1).
There was a significant main effect for race stage (P \ 0.01)
with no significant difference between the groups (P = 0.82)
and no significant supplementation group 9 race stage
interaction (P = 0.85). [CK] increased significantly from
baseline levels, peaking on stage 3 (PLA 1,067.9 ±
495.1 U L-1 vs. CHO–PRO 902.9 ± 186.3 U L-1), and
remained significantly elevated until the end of the race i.e.
stage 8 (Fig. 2).
For palpated muscle soreness, there were significant
main effects for time (P \ 0.01) and race stage (P \ 0.01),
and a significant time 9 race stage interaction (P \ 0.01).
There were no significant differences between supple-
mentation groups (P = 0.44) and no significant group
interactions. Palpated muscle soreness increased signifi-
cantly from baseline values to the end of race stage 1
(P \ 0.01), remained elevated at all measurement points
throughout the remainder of the race and increased sig-
nificantly from pre- to post-exercise for each individual
stage (Fig. 3).
Blood [glucose]
A repeated-measures ANOVA revealed a significant main
effect for time (P \ 0.01) with no significant differences
between supplementation groups (P = 0.40) and no
Race Stage (Sample Time)
Fig. 2 Creatine kinase concentration pre- and post-exercise in the
PLA and CHO–PRO groups. Data are presented as the mean
(circles) ± S.E.M. (error bars); significant difference from Stage 1
(pre-exercise)
significant interactions. There was a significant increase in
blood [glucose] pre- to post-exercise on both race stage 1
and 8 (both P \ 0.01), with no significant differences
between the groups (Fig. 4).
Urine osmolality
There was a significant main effect for supplementation
group (P = 0.04) but not for time (P = 0.96) or race stage
(P = 0.09). There were no significant interactions with the
exception of race stage 9 group (P = 0.04). Post hoc
analysis revealed that urine osmolality was significantly
higher post-exercise in CHO–PRO compared to PLA on
race stages 4 (P = 0.03, 95% CI 27–406 mosmol kg-1), 5
(P \ 0.01, 95% CI 109–461 mosmol kg-1), 6 (P = 0.01,
95% CI 62–394 mosmol kg-1), 7 (P = 0.04, 95% CI
2–305 mosmol kg-1) and 8 (P = 0.03, 95% CI
29–486 mosmol kg-1) (Fig. 5).
Tympanic membrane temperature
A repeated-measures ANOVA revealed that there was a
significant main effect for race stage (P \ 0.01) and sup-
plementation groups (P = 0.01) but no significant race
stage 9 group interaction (P = 0.98) (Fig. 6). Post hoc
analysis revealed that the rise in Tty from pre- to post-exer-
cise was significantly higher in PLA compared to CHO–PRO
on race stages 5 (P = 0.01, 95% CI 0.2–0.9°C), 6 (P = 0.03,
95% CI 0.1–0.8°C) and 7 (P = 0.01, 95% CI 0.2–1.1°C)
with a tendency observed on stage 2 (P = 0.07). Mean pre-
exercise Tty across all 8 stages was 37.0 ± 0.1°C in the PLA
group and 36.9 ± 0.1°C in the CHO–PRO group, rising to

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 Eur J Appl Physiol

Fig. 3 Palpated muscle 7

†
soreness pre- and post-exercise
in the PLA and CHO–PRO *
groups. Data are presented as 6
*
the mean (circles) ± SEM

Palpated Muscle Soreness

*
(error bars); significant 5 * * *
*
difference from stage 1 (pre-
exercise); *significant 4 *
difference from pre-exercise

2
PLA
1 CHO-PRO

0
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
(pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post)
Race Stage (Sample Time)

Pre-Exercise Post-Exercise Exercise performance

Day 1 Day 8
The CHO–PRO group completed the 8 stages significantly
9 *
* quicker than the PLA group (2,277 ± 127 vs.
Blood [Glucose] mmol⋅L-1

8
* * 2,592 ± 68 min; P = 0.02, 95% CI 63–618 min)
7
6
(Table 4). There were no significant differences in com-
5 pletion time for race stage 1 between groups (P = 0.09)
4 but the CHO–PRO group completed each subsequent stage
3 significantly faster than the PLA group (P \ 0.05).
2
1
0 Discussion
PLA CHO-PRO PLA CHO-PRO

Fig. 4 Blood [glucose] pre- and post-exercise on race stages 1 and 8 The aim of the present study was to investigate whether the
in the PLA and CHO–PRO groups. Data are presented as the mean addition of PRO to a standard CHO supplement would
(columns) ± SEM (error bars); *significant difference from pre- influence thermoregulation, muscle damage and exercise
exercise sample
performance during an intense, prolonged, multi-day
cycling competition in a hot environment. The results
demonstrate that the group provided with combined CHO–
38.5 ± 0.1°C in PLA and 38.1 ± 0.1°C in CHO–PRO by PRO supplements maintained their body mass during the
post-exercise. competition (Fig. 1), had an attenuated rise in Tty (Fig. 6)
and completed the race 12% quicker than those supple-
Heart rate mented with CHO alone (Table 4). No differences were
observed in either quantitative ([CK], Fig. 2) or subjective
There was a significant main effect for race stage (P = 0.04, (palpated muscle soreness score, Fig. 3) measurements of
P = 0.02) but no effect for supplementation group muscle damage between the two supplementation groups.
(P = 0.16, P = 0.24) and no significant race stage 9 group The differences between groups may, at least in part, be
interaction (P = 0.35, P = 0.33), for mean HR and HRpeak, due to an increased caloric delivery during exercise
respectively (Fig. 7). Post hoc analysis revealed both mean resulting from the additional PRO and a PRO-mediated
(PLA 154 ± 3 beats min-1; CHO–PRO 149 ± 4 beats expansion of body water compartments. To our knowledge,
min-1) and HRpeak (PLA 181 ± 1 beats min-1; CHO–PRO this is the first report to investigate the effects of CHO–
174 ± 1 beats min-1) were significantly higher on race PRO supplementation on physiological responses and
stage 1 compared to all other stages with no differences field-based competitive performance. The findings there-
observed between those remaining stages i.e. stages 2–8. fore lend support to the gathering body of evidence that

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Eur J Appl Physiol

Fig. 5 Urine osmolality pre- 1200

and post-exercise in the PLA PLA
and CHO–PRO groups. Data are CHO-PRO
1100
presented as the mean
(circles) ± SEM (error bars);

Urine Osmolality (mosmol·kg -1)

*significant difference between 1000 *
* * *
PLA and CHO–PRO groups *

900

800

700

600

500

400
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8
(pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post) (pre) (post)
Race Stage (Sample Time)

2.5 190
Rise in Tympanic Membrane Temperature (o C)

CHO-PRO Mean HR
PLA
* PLA Mean HR
180
CHO-PRO CHO-PRO Peak HR
2
PLA Peak HR
Heart Rate (beats·min -1)

* 170

1.5 * *
160
*

1 150

140
0.5

130
0
1 2 3 4 5 6 7 8 120
Race Stage 1 2 3 4 5 6 7 8
Race Stage
Fig. 6 Rise in tympanic membrane temperature from pre- to post-
Fig. 7 Mean (circles) and peak (triangles) heart rate in the PLA and
exercise in the PLA and CHO–PRO groups. Data are presented as the
CHO–PRO groups. Data are presented as the mean (circles and
mean (circles) ± SEM (error bars); *significant difference between
triangles) ± SEM (error bars); *significant difference between PLA
PLA and CHO–PRO groups
and CHO–PRO groups

combined CHO–PRO supplementation is advantageous for are likely to account for the differences observed. For
repeated, prolonged exercise performance in the heat. example, CHO–PRO beverages in the studies by Romano-
The observation in the present study that combined Ely et al. (2006) and Toone and Betts (2010) had a 7.5 and
CHO–PRO supplementation enhanced exercise perfor- 6.8% CHO content, respectively, compared with the 9%
mance compared with CHO alone is consistent with most CHO content of the isocaloric control group. Given the
(Berardi et al. 2008; Ivy et al. 2003; Saunders et al. 2004, well established concept that premature fatigue during
2007, 2009; Williams et al. 2003), but not all (Romano-Ely endurance exercise in temperate conditions is associated
et al. 2006; Toone and Betts 2010; van Essen and Gibala with CHO depletion (Coggan and Coyle 1987; Coyle et al.
2006), previous studies. Moreover, this study is the first to 1983), it is unsurprising that exogenous CHO supplemen-
demonstrate the ergogenic benefits of CHO–PRO supple- tation has been shown almost unequivocally to improve
mentation in a true competitive setting. Varying method- exercise performance in temperate conditions (Coggan and
ologies across studies including experimental design, the Coyle 1987; Coyle et al. 1983, 1986). Therefore, the fact
method of measuring exercise performance and whether that exercise performance was either unaffected (Romano-
the control group was matched for caloric or CHO content, Ely et al. 2006) or negatively affected (Toone and Betts

123

Table 4 Completion time (min) for each race stage of the two groups
Placebo CHO–PRO
group (n = 10) group (n = 13)
Stage 1 290 ± 27 278 ± 40
Stage 2 324 ± 34 300 ± 65*
Stage 3 326 ± 22 278 ± 43*
Stage 4 316 ± 30 259 ± 76*
Stage 5 349 ± 40 311 ± 72*
Stage 6 355 ± 31 314 ± 74*
Stage 7 361 ± 26 316 ± 71*
Stage 8 273 ± 50 223 ± 63*
Total 2,592 ± 68 2,277 ± 127*
Data presented as mean ± SEM
* Significant difference (P \ 0.05) between groups
2010) when the CHO content of the CHO–PRO supple-
ments was significantly lower than that in the control group
is somewhat intuitive. In the present study the CHO–PRO
and CHO-only supplements were matched for CHO con-
tent (*7.5%). Consequently, although liquid and solid
ingestion rates were not different between groups
(Table 3), the additional PRO content in the CHO–PRO
supplements ensured the rate of caloric intake was signif-
icantly higher in the CHO–PRO group (PLA
230 ± 10 kcal h-1 vs. CHO–PRO 300 ± 27 kcal h-1,
Table 3). It is therefore possible that the superior exercise
performance observed in CHO–PRO was mediated by an
increased delivery of energy during the exercise period.
Data from Colombani et al. (1999) does indeed demon-
strate that PRO added to CHO can be utilised as an energy
substrate during endurance exercise. The significant
decline in body mass observed in the PLA group from the
1st to the 8th stage and the small but non-significant
increase in body mass observed in the CHO–PRO group
(Fig. 1) lends some support to this theory. However,
Saunders et al. (2004) have suggested that while this
mechanism should not be entirely discounted, the relatively
small difference in caloric intake is unlikely to explain the
large improvements in exercise performance observed in
other studies.
Alternatively, it has been suggested that recovery from a
previously fatiguing bout of exercise is enhanced by CHO–
PRO supplementation and may explain the observed
improvement in subsequent exercise performance (Berardi
et al. 2008; Ivy et al. 2002; Saunders et al. 2004, 2007;
Williams et al. 2003). Several studies have demonstrated
either an increase in muscle glycogen restoration mediated
by augmented insulin concentration (Berardi et al. 2008;
Ivy et al. 2002; Williams et al. 2003) or a reduction in
markers of muscle damage (Cockburn et al. 2008;
Romano-Ely et al. 2006; Saunders et al. 2004, 2007, 2009)
123
Eur J Appl Physiol
when CHO–PRO is ingested during exercise or recovery,
compared to CHO alone. Enhancing muscular recovery
through CHO–PRO ingestion would seem particularly
pertinent in the present study where competitors were
required to perform repeated bouts of relatively high
intensity, long duration exercise. Indeed, despite no dif-
ferences in completion time for the first race stage, subjects
in the CHO–PRO group completed all subsequent stages
significantly faster than the PLA group (Table 4). This is
certainly consistent with the theory that exercise perfor-
mance may be enhanced by an improvement in muscle
glycogen restoration mediated by the CHO–PRO supple-
mentation. Though we did not measure muscle glycogen
storage here, and thus the effects of CHO–PRO on glyco-
gen resynthesis cannot be elucidated, it should be noted
that several studies have found no differences in muscle
glycogen storage between CHO–PRO and CHO-only sup-
plements (Carrithers et al. 2000; Millard-Stafford et al.
2005; Van Hall et al. 2000). Furthermore, Burke et al.
(2004) have suggested that feeding a large quantity of CHO
at frequent intervals (as in the present study) negates the
beneficial effect of additional PRO on glycogen storage.
No differences were observed in either [CK] or palpated
muscle soreness between CHO–PRO and PLA groups
suggesting that additional PRO ingestion during exercise
does not affect concurrent or indeed subsequent muscle
damage. This is in direct contrast to the majority of pre-
vious studies which have reported significant reductions in
[CK] (Cockburn et al. 2008; Saunders et al. 2004), palpated
muscle soreness (Millard-Stafford et al. 2005) or both
(Romano-Ely et al. 2006; Saunders et al. 2009) following
supplementation with CHO–PRO. The putative mechanism
underlying these reductions in muscle damage following
CHO–PRO compared to CHO-only supplementation has
been suggested to be an increased rate of PRO synthesis
and repair (Saunders et al. 2004). The reason why CHO–
PRO supplementation did not attenuate markers of muscle
damage in the present study, therefore, is unclear, but
perhaps relates to the ad libitum ingestion of additional
PRO in both groups outwith the race stages (Table 3). That
is, participants in both groups were given free access to
food at evening meals and although there were no signifi-
cant differences in either the energy content or the com-
position of the diet between groups, as would be expected,
these meals contained significant PRO content (Table 3).
Thus, it appears that when PRO and CHO intake following
exercise is unrestricted, additional PRO ingestion during
exercise may not significantly reduce muscle damage
compared to CHO supplementation alone. Alternatively,
the higher work rates employed by the CHO–PRO group,
as demonstrated in their significantly faster race comple-
tion time, may result in an increased amount of muscle
damage that is counteracted by any PRO-induced
Eur J Appl Physiol
reductions i.e. due to increased synthesis and repair. It is
also feasible that the large variability associated with [CK]
measurements (Millard-Stafford et al. 2005; Saunders et al.
2004) and the subjectivity of muscle soreness ratings meant
that these indirect markers lacked the sensitivity to identify
any true differences in muscle damage.
Though no differences in muscle damage were observed
between groups, one of the primary findings of interest was
a significant attenuation in the tympanic membrane tem-
perature from pre- to post-exercise in CHO–PRO compared
to the PLA group (Fig. 6). Even with the significantly
faster race completion time, the mean rise in tympanic
membrane temperature after each stage was 0.43 ± 0.04°C
less in CHO–PRO than PLA. Furthermore, both mean HR
and HRpeak were lower during each stage in CHO–PRO
versus PLA (Fig. 7), although these differences were not
statistically significant. As there were no differences in age,
body mass, height or predicted aerobic capacity between
the groups (Table 1) the reduction in thermal strain is
unlikely to be related to disparities in anthropometric or
fitness characteristics. Goto et al. (2007) and Okazaki et al.
(2009) have recently reported that combined CHO–PRO
supplementation during a period of aerobic training resul-
ted in a significant improvement in the cardiovascular and
thermoregulatory capacities of both young and old healthy
males. The authors suggested that these effects were
mediated by a CHO–PRO-induced increase in albumin
synthesis and plasma albumin content, which resulted in a
fluid shift from the interstitial to the intravascular space and
an ensuing expansion in plasma volume. Indeed an increase
in plasma albumin has been shown to enhance the effective
colloid osmotic pressure gradient between the fluid com-
partments, resulting in a net fluid movement into the vas-
cular space to maintain plasma albumin concentration
(Convertino et al. 1980). Furthermore, Deschamps et al.
(1992) found that HR and core temperature responses were
attenuated and Luetkemeier and Thomas (1994) reported
that cycling performance was improved by more than 10%
following acute plasma volume expansion with either sal-
ine or dextran infusions. Conversely though, others have
failed to show any significant effect of plasma volume
expansion on HR, core temperature, skin blood flow or
even performance during exercise in the heat (Grant et al.
1997). Whilst plasma albumin concentration was not
directly measured in the present study, it remains a distinct
possibility that the additional PRO consumed by the CHO–
PRO group may have resulted in expansion of the plasma
volume which accordingly improved thermoregulatory
capacity and resulted in a smaller rise in tympanic tem-
perature during exercise.
There is, however, an alternative explanation for the
reduced level of thermal strain manifest in the CHO–PRO
group compared to the PLA group. Electrolyte
concentrations in both liquid and solid supplements were
higher in the CHO–PRO group than in PLA. Maughan
and Leiper (1995) have demonstrated that there is a direct
relationship between the amount of Na? in a rehydration
solution and the fraction of fluid retained. Therefore, the
higher sodium content of the CHO–PRO supplements in
the present study may have resulted in a larger volume of
fluid being retained compared to the PLA supplements
and so may offer a significant thermal advantage. The
significantly higher urine osmolality in the CHO–PRO
group during race stages 4–8 (Fig. 5), despite no differ-
ence in fluid ingestion rates (Table 3), would seem to
suggest that subjects in the PLA group did indeed excrete
a greater proportion of the ingested fluid. However, this
may simply be caused by an increased excretion rate of
N2 in the CHO–PRO group following the breakdown of
excess PRO (Dangin et al. 2001). Alternatively, the higher
urine osmolality in the CHO–PRO group may stem from
the fact that the addition of PRO to a standard CHO
supplement will increase its energy density which can
have a profound effect on gastric emptying time (Mau-
ghan et al. 2004). Energy density is reported to be one of
the primary factors regulating the rate at which ingested
fluid empties from the stomach, with more energy dense
solutions taking significantly longer to empty (Maughan
et al. 2004). Therefore, the higher urine osmolality in the
CHO–PRO group may be caused by a relative delay in
fluid transit time between ingestion and excretion in the
PLA group.
It is acknowledged that due to the potential impact of
several uncontrollable confounding factors the findings of
the present study are not without limitation. A measure-
ment of baseline performance and physiological responses
to exercise in the two groups of subjects would have been
advantageous, but was not possible given the time and
logistical constraints of the study. Similarly, a randomised
cross-over design would have strengthened confidence in
the observed results. However, these limitations are bal-
anced with the ecological validity offered by examining the
effects of CHO–PRO supplementation on competitive
exercise performance. All too often the effects of nutri-
tional interventions are examined under tightly controlled
laboratory conditions, which do not provide meaningful
insight as to whether an ergogenic effect can be obtained
during actual competition.
Conclusion
In the present study, combined CHO–PRO supplementa-
tion as opposed to CHO alone during a prolonged, intense
8-day mountain bike race in a hot environment was
effective in preventing body mass loss, reducing tympanic
123

membrane temperature rise and improving exercise
performance. These effects may be mediated by a PRO-
induced increase in substrate availability and/or improve-
ments in thermoregulatory capacity; the exact mechanism
remains unknown, however. The physiological and per-
formance benefits reported here suggest that CHO–PRO
supplementation may be advantageous for athletes exer-
cising in hot conditions over prolonged periods of time.
Furthermore, the addition of PRO did not reduce ad libitum
ingestion rates, suggesting palatability of the beverages
was not negatively affected. However, further laboratory-
based research which controls for confounding and extra-
neous factors should be completed to further explore the
ergogenic potential of CHO–PRO supplementation during
exercise in the heat.
Acknowledgments This study was supported in part by High5 Ltd,
UK.
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