Effect of Increased Dietary Protein on

Tolerance to Intensified Training

OLIVER C. WITARD, SARAH R. JACKMAN, ARIE K. KIES, ASKER E. JEUKENDRUP, and KEVIN D. TIPTON
Human Performance Laboratory, Exercise Metabolism Research Group, School of Sport and Exercise Sciences,
The University of Birmingham, Edgbaston, UNITED KINGDOM

ABSTRACT
WITARD, O. C., S. R. JACKMAN, A. K. KIES, A. E. JEUKENDRUP, and K. D. TIPTON. Effect of Increased Dietary Protein on
Tolerance to Intensified Training. Med. Sci. Sports Exerc., Vol. 43, No. 4, pp. 598–607, 2011. Purpose: The purpose of the present study
was to examine the effect of increased protein intake on short-term decrements in endurance performance during a block of high-intensity
training. Methods: Trained male cyclists (V̇O2max = 64.2 T 6.5 mLIkgj1Iminj1) completed two 3-wk trials both divided equally into
normal (NOR), intensified (INT), and recovery (REC) training. In a counterbalanced crossover experimental design, cyclists received
BASIC SCIENCES

either a high-protein (PRO; 3 g proteinIkgj1 body mass (BM)Idj1) or a normal diet (CON; 1.5 g proteinIkgj1 BMIdj1) during INT and
REC. Dietary carbohydrate content remained constant at 6 gIkgj1 BMIdj1. Energy balance was maintained during each training week.
Endurance performance was assessed with a V̇O2max test and a preloaded time trial. Alterations in blood metabolite responses to exercise
were measured at rest, during, and after exercise. Cyclists completed the Daily Analysis of Life Demands for Athletes (DALDA)
questionnaire each day. Results: Increased dietary protein intake led to a possible attenuation (4.3%; 90% confidence limits
/5.4%) in
the decrement in time trial performance after a block of high-intensity training compared with NOR (PRO = 2639 T 350 s; CON = 2555 T
313 s). Restoration of endurance performance during recovery training possibly benefited (2.0%;
/4.9%) from additional protein
intake. Frequency of symptoms of stress described as ‘‘worse than normal’’ reported after a block of high-intensity training was very
likely (97%) attenuated (17; T11 AUC of ‘‘a’’ scores part B, DALDA for INT + REC) by increasing the protein content of the diet.
No discernable changes in blood metabolite concentrations were observed in PRO. Conclusions: Additional protein intake reduced
symptoms of psychological stress and may result in a worthwhile amelioration of the performance decline experienced during a
block of high-intensity training. Key Words: OVERLOAD TRAINING, PROTEIN FEEDING, TIME TRIAL PERFORMANCE,
PSYCHOLOGICAL STATUS

I
n an attempt to improve endurance performance, ath-
letes often incorporate periods of high training vol-
ume, combined with limited recovery time, into carefully
planned training regimens (11). A disproportionate balance
between training impulse and recovery is likely to result in
an accumulation of fatigue, together with perturbations in
biological functioning and psychological status (38). Such
an imbalance in homeostasis with intensified training ulti-
mately results in an acute impairment in exercise capacity
(14) and/or performance (2,12,20).
Endurance athletes interested in enhancing recovery from
each intense bout of exercise commonly use nutrition as
a tool (15). Carbohydrate (CHO) feeding has been dem-
Address for correspondence: Kevin D. Tipton, Ph.D., Exercise Metabolism
Research Group, School of Sport and Exercise Sciences, University of
Birmingham, Edgbaston, Birmingham, B15 2TT, UK; E-mail: k.d.tipton@
bham.ac.uk.
Submitted for publication December 2009.
Accepted for publication August 2010.
0195-9131/11/4304-0598/0
MEDICINE & SCIENCE IN SPORTS & EXERCISEÒ
Copyright Ó 2011 by the American College of Sports Medicine
DOI: 10.1249/MSS.0b013e3181f684c9
onstrated to attenuate short-term decrements in endurance
performance/capacity after intensified periods of training
(2,14). Supplementing a habitual diet for 8 d with CHO be-
fore, during, and after (6%, 6%, and 20% CHO solution, re-
spectively) each intense training session (14) or administering
a high-CHO diet (8.5 gIkgj1 body mass (BM)Idj1) compared
with a moderate-CHO diet (5.4 gIkgj1 BMIdj1) (2) before
and during periods of increased training volume resulted in an
attenuated impairment in endurance performance. Better
maintenance of endurance performance and mood state with
CHO feeding during intensified training was associated with
attenuated impairments in the stress hormone response to
exercise (14) and maintained rates of muscle glycogenoly-
sis (2). In both studies (2,14), protein content of the diet
(È1.9 gIkgj1 BMIdj1) remained constant. CHO has received
considerable attention, but despite some evidence, the effect
of protein feeding on exercise recovery from an intensified
period of training is yet to be investigated.
In a short-term setting, the importance of protein intake
during the immediate few hours after endurance exercise has
recently received a great deal of attention. The addition of
protein to a CHO-rich drink, administered during and after
exercise, was reported to improve the endurance capacity of
subsequent exercise compared with a CHO-matched but
nonisoenergetic drink (27,33). Possible mediators for the

598

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improved recovery with additional protein intake include the
amelioration of markers of muscle damage, the modulation
of postexercise protein balance (21,22), and the initiation of
insulin-elicited accelerated rates of glycogen repletion after
exercise (39). Moreover, the potential for a high-protein diet
to impart a small but beneficial increase in energy substrate
availability via gluconeogenic pathways during endurance-
type activities cannot be ruled out. Thus, the modulation
of multiple physiological responses has been proposed to
explain the potentially beneficial role of protein nutrition for
optimizing recovery from a strenuous acute bout of endur-
ance exercise.
Given that there may be some benefit of acute ingestion of
protein for endurance exercise recovery, it follows that long-
term protein ingestion may also be beneficial; however, to
date, no study has directly examined the effect of long-term
protein feeding on endurance performance in the context of
a block of high-intensity training. In a recent field-based
study (10), U.S. Marine recruits reported a better mainte-
nance of general health and a reduction in local muscular
disruptions when protein was added to nutritional sup-
plements consumed immediately after each training session of
a basic 54-d training period compared with those without
protein (10). These data provide preliminary evidence that the
beneficial role of protein supplementation for exercise re-
covery observed in short-term–based studies may be extrap-
olated to the long-term effect of protein feeding on recovery
from a period of intensified training. However, a more sys-
tematic examination of the response of repeated bouts of ex-
ercise to long-term protein feeding is yet to be undertaken.
The purpose of this proof-of-concept study was to exam-
ine the effect of additional dietary protein intake during a
short-term period of intensified training on subsequent en-
durance performance. We hypothesized that trained cyclists
would experience an improved tolerance to the stress of
increased training volume (i.e., fewer perturbations in psy-
chological mood state and markers of endocrine function)
when consuming a high-protein diet during and after intensi-
fied training, ultimately resulting in the better maintenance
of endurance performance.
METHODS
Subjects
Eight endurance-trained cyclists (age = 27 T 8 yr, BM =
73 T 7 kg; maximal oxygen uptake (V̇O2max) = 64.2 T
6.5 mLIkgj1Iminj1, maximal power output (Wmax) = 372 T
21 W) were recruited to participate in this study. All cyclists
had a training history of at least 5 yr. Before participation,
the health status of each participant was assessed using a
general health questionnaire. A detailed explanation of prac-
tical details, associated risks, and required commitment
applicable to the present study preceded the attainment of
written informed consent. Participants were reminded of their
right to withdraw from the study at anytime without provision
PROTEIN, INTENSE TRAINING, AND PERFORMANCE
Copyright © 2011 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
of reason. The protocol was approved by the research ethics
committee of the School of Sport and Exercise Sciences,
University of Birmingham, United Kingdom.
Experimental Design
This study was designed to determine whether increas-
ing dietary protein intake leads to a meaningful attenuation
of the short-term decrement in endurance performance, ele-
vated mood disturbance, and perturbations in biological func-
tioning induced by a block of high-intensity training. Each
participant completed two trials, both consisting of a 3-wk
period of quantified training. Sequentially, training periods
were divided into 1 wk of normal training (NOR), 1 wk of
intensified training (INT), and 1 wk of recovery training
(REC). Endurance performance was assessed by a V̇O2max
test and preloaded time trial on days 6 and 7, respectively,
of each training week. Identical diets were administered dur-
BASIC SCIENCES
ing NOR in both conditions. In a counterbalanced crossover
experimental design, participants received either a diet con-
sisting of a high-protein intake (PRO) or a normal protein
intake (CON) during INT and REC. Trials were separated by
at least a 2-wk washout period in which participants resumed
their normal training volume and diet.
Preliminary Testing
Wmax, V̇O2max, submaximal V̇O2 (determined by expired
gas collections at each increment of V̇O2max test), and
maximal HR (HRmax) were determined within the 7 d before
commencing each experimental trial, as previously described
(14). All participants completed a familiarization time trial
within 7 d before commencement of the study.
Dietary Analysis
In the week preceding the first trial, a 3-d weighed food
record was completed. Participants were asked to record accu-
rately all food items they consumed during two weekdays and
one weekend day. Diets were analyzed using Internet-based
nutrition analysis software (www.weightlossresources.co.uk).
The habitual daily energy intake of the subjects was 2882 T
172 kcal, and the habitual diet consisted of the following
macronutrient intake: protein = 1.6 T 0.1 gIkgj1 BMIdj1,
CHO = 5.4 T 0.3 gIkgj1 BMIdj1, and fat = 1.3 T
0.1 gIkgj1 BMIdj1.
Experimental Trial Periods
Training quantification. In an attempt to monitor train-
ing as accurately as possible and ultimately standardize
training impulse between trials, each participant received a
cyclists’ handbook. On a daily basis, cyclists completed a
training diary (time of day training session was started, dura-
tion of training (min), average and maximal HR (beatsIminj1)
for training sessions, location of training, weather conditions,
and distance covered (km)) and an assessment of well-being
(morning resting HR (RHR; beatsIminj1), sleep rating (Likert
Medicine & Science in Sports & Exercised 599
 scale, 1–10), stress rating (Likert scale, 1–10), fatigue rating
(Likert scale, 1–10), total sleep (h), and morning BM (kg)).
Upon waking, RHR was recorded as the average HR during a
4-min period. On day 6 of each training week, cyclists wore
their HR monitors throughout the night (from going to bed
at night to getting up in the morning) to record an average
sleeping HR (SHR).
Athletes were equipped with a downloadable HR monitor
(Polar Vantage NV, Kempele, Finland) for the duration of
each trial to monitor individual training sessions. Training
zones were calculated in accordance with British Cycling
guidelines (1), using lactate threshold (LT) and HR values
derived from the preliminary V̇O2max test. Five training zones
were defined as follows: zone 1 (Z1) = G70%HRmax, zone
2 (Z2) = 70%–80%HRmax, zone 3 (Z3) = 80%–90%HRmax,
zone 4 (Z4) = 90%–95%HRmax, and zone 5 (Z5) =
995%HRmax. During NOR, participants completed their
BASIC SCIENCES
usual volume and type of training. Athletes were required to
train on a daily basis during INT, which typically consisted of
a combination of high-intensity interval sessions above LT,
lasting between 2 and 3 h, and long, continuous rides, usually
between 4 and 5 h in duration. Consequently, time spent in
Z3–Z5 were increased during INT compared with NOR in
both trials. During REC, training volume was reduced to
È60% relative to NOR in both dietary conditions.
Dietary control. Diet was controlled for the duration of
the study with all food prepared by investigators in the
Metabolic Research Kitchen. Five set menus were carefully
composed using the Internet-based nutrition analysis soft-
ware (www.weightlossresources.co.uk). Food and drinks
were provided as three main meals together with a collection
of snacks. Participants were asked to refrain from consum-
ing alcohol and caffeine for the duration of the study.
For each dietary period, participants were fed with the aim
to maintain energy balance. The summation of basal meta-
bolic rate (kcalIdj1) (ambulating status), predicted using the
Harris Benedict equation and estimated energy expenditure
in cycling activity (25), was used to estimate daily energy
requirements of cyclists. Total energy intake was adjusted
on a daily basis to ensure weight stability. Energy intake
was matched between dietary conditions during NOR
(PRO/CON = 3711 T 456 kcalIdj1), INT (PRO = 4409 T
432 kcalIdj1; CON = 4410 T 437 kcalIdj1), and REC
(PRO = 3096 T 503 kcalIdj1; CON = 3096 T 432 kcalIdj1).
The macronutrient composition of the diet during NOR
was equivalent to 1.5 g of proteinIkgj1 BMIdj1 and 6 g of
CHOIkgj1 BMIdj1, with the remainder of energy derived
from fat. Hence, for 7 d before a block of high-intensity
training, dietary intake and composition were identical
between trials. In CON, diet composition remained the same
as NOR in both INT and REC, with energy intake modified
to match daily changes in energy expenditure. In PRO,
during both INT and REC, protein intake was doubled to
3 gIkgj1 BMIdj1. As intended, dietary protein intake was
significantly greater in PRO compared with CON during
INT (PRO = 210 T 17 gIdj1; CON = 106 T 9 gIdj1) and
600 Official Journal of the American College of Sports Medicine
Copyright © 2011 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
REC (PRO = 210 T 18 gIdj1; CON = 105 T 11 gIdj1). The
CHO content of the diet was held to 6 gIkgj1 BMIdj1,
with the remainder of energy derived from fat. Intake of fat
was greater in CON compared with PRO during both INT
(PRO = 185 T 24 gIdj1; CON = 239 T 30 gIdj1) and REC
(PRO = 122 T 54 gIdj1; CON = 167 T 52 gIdj1).
In both dietary conditions, CHO-rich fruit smoothies were
consumed daily during INT and REC. In PRO, 60 g (3
20 g)
of casein protein isolate (DSM Food Specialties, Delft,
The Netherlands) was added to a fruit smoothie to assist the
attainment of increased dietary protein intake. In CON, cream
was added to ensure the desired fat intake could be attained.
Participants were advised to ingest smoothies on immediate
completion of training sessions. All participants confirmed
their awareness of which dietary intervention was being
administered during each trial.
Performance Tests
Excluding preliminary testing, subjects completed a total
of six V̇O2max tests and six time trials to assess maximal
power output and endurance performance, respectively.
V̇O2max tests and time trials were always performed in the
morning (start of exercise between 06:30 and 08:00 a.m.)
under similar environmental conditions (È19-C and È55%
relative humidity) and in a fasted state (910 h). The same
incremental (workload increased by 35 W at 3-min intervals
until volitional exhaustion) maximal cycle ergometer V̇O2max
test protocol was performed during experimental trial periods
as previously described (14).
Time trial. To determine endurance performance, a pre-
loaded (120 min of submaximal exercise at 50% Wmax) time
trial that lasted approximately 45 min (19) was performed
on day 7 of each training week. Previously described general
procedures for performance testing were adhered to (8,19).
Briefly, on arrival at the laboratory, subjects were fitted with
an HR monitor, and a Teflon catheter was inserted into a
forearm vein. After a 10-min rest period, a baseline blood
sample was drawn. Subjects cycled for 120 min at 50% Wmax
with the electromagnetically braked ergometer set in the
hyperbolic (cadence-independent) mode so that work rate
was independent of pedaling rate. At regular time points
(20, 40, 60, 80, and 100 min), blood samples (È15 mL) were
drawn, HR was recorded, and RPE, using the modified Borg
scale (4), were obtained. These measurements were repeated
immediately after completion of the 120-min submaximal
exercise preload (SM). The ergometer was then adjusted to
the cadence-dependent (linear) mode. Cyclists commenced
the simulated time trial and were required to complete a set
amount of work (670 T 52 kJ) as fast as possible. The total
amount of work to be performed was calculated using the
following formula (19):
total work ðJÞ ¼ 0:7Wmax
2700 s ½1
The linear factor was individually adjusted, so that 70% Wmax
was obtained when the subject pedaled at their preferred
cadence. The only information available to the subjects
http://www.acsm-msse.org
during the time trial was elapsed work and percentage of
work performed (i.e., 0% at the start and 100% on comple-
tion). Further blood samples were collected at immediate
cessation of time trial and after 1 h of passive recovery.
Blood Analyses
Blood samples were collected into prechilled vacutainers
containing K3EDTA or lithium heparin (Becton Dickinson,
Franklin Lakes, NJ). Whole blood was immediately placed on
ice until centrifugation at 3000 rpm for 10 min at 4-C, within
2 h of collection. Plasma was stored at j80-C until further
analysis. Enzymatic analysis for plasma lactate (lactic acid;
ABX Diagnostics, Chicksands, UK) and urea concentrations
(urea; ABX Diagnostics) were determined in duplicate using
a semiautomated analyzer (COBAS MIRA S-plus; ABX
Diagnostics). Commercially available sandwich ELISA kits
were used to determine plasma cortisol (cortisol; IDS, Tyne
and Wear, UK), testosterone (testosterone; IDS), and epi-
nephrine (CAT-COMBO; IDS) concentrations. In each case,
plates were read in duplicate on a Labsystems Original Mul-
tiskan MS at selected wavelengths (cortisol and testosterone =
450 nm; epinephrine = 405 nm). Reported sensitivities of
ELISA were 2.5 ngImLj1, 0.083 ngImLj1, and 10 pgImLj1
for cortisol, testosterone, and epinephrine, respectively. Intra-
assay variations were calculated for cortisol (7%), testoster-
one (8%), and epinephrine (9%). Plasma urea concentrations
were determined at all time points during the time trial. Hor-
mone concentrations were measured at baseline, following
the submaximal exercise preload and immediately after the
time trial. In addition, plasma cortisol was also measured af-
ter 1 h of passive recovery. Blood samples collected during
each stage of the V̇O2max test were analyzed for plasma lactate
concentrations.
Questionnaires
Daily analysis of life demands for athletes. Par-
ticipants completed the Daily Analysis of Life Demands for
Athletes (DALDA) questionnaire (31) on a daily basis to
monitor psychological status. The DALDA is divided into
parts A and B, which represent the sources and manifesta-
tion of stress in the form of signs and symptoms, respectively.
For both parts, the number of items marked as ‘‘worse than
normal’’ (‘‘a’’ scores) were tallied and reported.
POMS-65. On day 7 of each training week, before
exercise, participants completed a 65-item version of the
POMS-65 questionnaire (26). POMS-65 items are divided
into six categories: tension, depression, anger, vigor, fatigue,
and confusion. Global mood state (GMS) was calculated as
the sum of all negative categories minus the score for vigor,
plus 100.
Data Presentation and Statistical Analysis
All data are expressed as means T SD. A two-way (dietary
condition and week per day) ANOVA with repeated mea-
PROTEIN, INTENSE TRAINING, AND PERFORMANCE
Copyright © 2011 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
sures was used as the statistical model to determine differ-
ences between dietary conditions, unless otherwise stated.
Simple contrasts between diet (two levels: PRO and CON)
and week (two levels: NOR and INT, NOR and REC) were
selected to generate P values. Plasma urea concentration
measured during the time trial and ‘‘a’’ scores on part B
of DALDA were expressed as AUC (baseline set as resting
values measured during NOR of corresponding dietary con-
dition). Statistical analysis was performed using Statistical
Package for Social Sciences (SPSS) 15.0 for Windows (SPSS,
Inc., Chicago, IL).
Mechanistic inferences about the population values of
statistics were made via magnitude-based precision of esti-
mation, as summarized recently (18). All data sets were
tested for normality using Kolmogorov–Smirnov normality
tests. If normality was violated, data were transformed
using natural log. Precision is presented as T90% confi-
BASIC SCIENCES
dence limits (CL) or
/ 90% CL for non–log-transformed
and log-transformed data sets, respectively. Effect sizes for
log-transformed data sets were calculated by dividing the
difference (CON j PRO) of the change (INT j NOR or
REC j NOR) in measured parameter by log-transformed
SD of CON. Thresholds were held constant at 0.2. For non–
log-transformed data sets, the threshold value for small was
calculated as 0.2
SD for CON. In this case, effect size
refers to the mean effect calculated as the difference between
PRO and CON for the change in value between NOR and
INT and NOR and REC.
The likelihood of the outcome being of beneficial,
detrimental, or trivial nature was also determined using
a published spreadsheet (17); likelihoods were qualified:
G1% almost certainly no chance, 1%–5% = very unlikely,
5%–25% = unlikely, 25%–75% = possible, 75%–95% =
likely, 95%–99% = very likely, and 999% = almost certain.
In the case where most (950%) of the uncertainty lies
between the threshold for a substantially positive and nega-
tive effect, the likelihood of the effect being trivial (negli-
gible) is qualified. Effects were described as unclear or
inconclusive if confidence intervals overlapped onto both
positive and negative values.
RESULTS
Quantification of Training
No clear effect of additional protein intake on weekly
training volume was observed during NOR (G1%), INT
(G1%), and REC (G1%). Additional protein intake did not
clearly affect time spent in each training zone during INT
(Z1: 1%, Z2: G1%, Z3: 2%, Z4: 5%, Z5: È1.3-fold increase)
and REC (Z1: 8%, Z2: 3%, Z3: 9%, Z4: G1%, Z5: G1%).
Time Trial Performance
Figure 1 displays time trial performance after NOR, INT,
and REC training in high-protein (PRO) and normal protein
(CON) trials. Increased dietary protein intake was unlikely
Medicine & Science in Sports & Exercised 601
 FIGURE 1—A, Time trial performance after normal (NOR), intensified (INT), and recovery (REC) training in high-protein (PRO) and normal
protein (CON) trials. Values are means T SD. B, Magnitude of decrement in time trial performance after intensified training (INT) and recovery (REC)
compared with normal training in high-protein (PRO) and normal protein (CON) trials. The greater the decrement in performance, the more positive
the log-transformed value. Data points refer to back-transformed means, which are fold effects. Dotted lines refer to 90% CI uncertainty bars.

(G1%) harmful and possibly (30%) attenuated (4.3%; (compared with NOR) was not clearly affected by additional
BASIC SCIENCES

CL
/5.4%) the decrement in time trial performance after protein.

a block of high-intensity training. Restoration of endurance
performance during recovery training possibly (48%) bene-
fited (2.0%;
/4.9%) and was most unlikely (G1%) harmed
by additional protein intake.
Physiological, Metabolic, and Biochemical
Responses
Maximal exercise responses. No clear effect of PRO
was observed for the decrement in Wmax after INT (PRO =
5% T 4%; CON = 6% T 4%) and REC (PRO = 1% T 3%;
CON = 1% T 3%) compared with NOR (PRO = 349 T 34 W;
CON = 357 T 27 W). Table 1 summarizes the intervention
effect of increased dietary protein intake on physiological,
metabolic, and biochemical responses to maximal exercise
after intensified training. The intervention effect of addi-
tional protein intake on modulated V̇O2max values measured
after INT (PRO = 4.36 T 0.11 LIminj1; CON = 4.19 T 0.73
LIminj1) and REC (PRO = 4.49 T 0.20 LIminj1; CON =
4.43 T 0.20 LIminj1) compared with NOR (PRO = 4.24 T
0.56 LIminj1; CON = 4.49 T 0.64 LIminj1) were very likely
(99.9%) trivial. No clear effects of additional protein intake
on maximal HR or maximal lactate concentrations were
detected after INT or REC. The decrement in maximal
HR (HRmax) (6 T 1 beatsIminj1) and lactate (LACmax)
(1.88 T 0.59 mmolILj1) responses to exercise after INT
Submaximal exercise and resting responses. No
intervention effect of diet on mean HR (PRO, mean
range = 127–128 beatsIminj1; CON, mean range = 125–128
beatsIminj1) and RPE (PRO, mean range = 12–14; CON,
mean range = 11–13) values recorded during the 120-min
preload of time trial, RHR (PRO, mean range = 51–52
beatsIminj1; CON, mean range = 50–53 beatsIminj1) or
SHR (PRO, mean range = 50–53 beatsIminj1; CON, mean
range = 50–51 beatsIminj1) was observed. Compared with
NOR, increasing dietary protein intake was very likely to
substantially increase the plasma urea response to exercise,
calculated as AUC, after both INT (PRO = 353; T164
mmolILj1; CON = 189; T76 mmolILj1) and REC (PRO =
300; T178 mmolILj1; CON = 150; T104 mmolILj1).
Hormonal responses. The response of peak stress
hormone concentrations to intensified training in PRO
and CON are displayed in Figure 2. No clear effect of PRO
was detected for the amelioration of lowered peak cortisol
concentrations after INT (7.1%;
/6.8%) and REC (7.6%;

/12.9%) compared with NOR. Similarly, the effect of
additional protein intake for the amelioration of lowered
peak epinephrine concentrations was unclear after INT (13%;

/129%) and REC (16%;
/53%) compared with NOR.
The intervention effect of additional protein intake on
modulated peak testosterone concentrations measured after

TABLE 1. Selected changes in maximal test variables over the course of the study period.
Diet Condition Outcome
Maximal Response PRO (Factor Effect)a CON (Factor Effect)b Mean Effectc;
/90% CL Qualitative Inference
Change from NOR after INT
V̇O2max (LIminj1) 1.03 (1.09) 0.93 (1.12) +11;
/1 Unclear
Maximal HR (beatsIminj1) 0.96 (1.04) 0.94 (1.04) +1;
/9 Unclear
Maximal lactate (mmolILj1) 0.67 (1.21) 0.81 (1.19) j17;
/2 Unclear
Change from NOR after REC
V̇O2max (LIminj1) 1.06 (1.11) 0.99 (1.14) +7;
/2 Unclear
Maximal HR (beatsIminj1) 1.01 (1.04) 0.99 (1.03) +1;
/78 Unclear
Maximal lactate (mmolILj1) 1.01 (1.22) 0.88 (1.31) +13;
/10 Unclear
a
Back-transformed mean difference after INT compared with NOR in PRO. Factor effects are shown in parentheses.
b
Back-transformed mean difference after REC compared with NOR in CON. Factor effects are shown in parentheses.
c
Mean effect of diet condition refers to back-transformed factor effects relative to the reference condition (NOR j INT; NOR j REC) between PRO and CON.

602 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org

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FIGURE 2—A, Peak cortisol and (B) peak epinephrine concentrations
after normal (NOR), intensified (INT), and recovery (REC) training in
high-protein (PRO) and normal protein (CON) conditions. Peak corti-
sol and epinephrine concentrations typically measured immediately
following the time trial.
INT (PRO = 7.0 T 1.7 ngImLj1; CON = 7.9 T 2.2 ngImLj1)
and REC (PRO = 6.6 T 1.4 ngImLj1; CON = 8.8 T
2.2 ngImLj1) compared with NOR (PRO = 7.5 T
2.2 ngImLj1; CON = 6.6 T 1.6 ngImLj1) were very likely
(100%) trivial. No clear effect of increased dietary protein
intake on testosterone–cortisol ratio was observed (data
not shown).
FIGURE 3—Daily reported number of ‘‘worse-than-normal’’ ‘‘a’’ scores (A) on part B (symptoms of stress) on the DALDA questionnaire during
normal (NOR), intensified (INT), and recovery (REC) training and ‘‘worse-than-normal’’ ‘‘a’’ scores (B) on part B (symptoms of stress) on the DALDA
questionnaire, expressed as AUC over INT and REC training weeks collectively. Cyclists completed the DALDA questionnaire on waking in a rested
state on a daily basis. Values are expressed as means T SD.
PROTEIN, INTENSE TRAINING, AND PERFORMANCE
Copyright © 2011 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
Mood State and Well-being
Daily analysis of life demands for athletes. Figure 3
displays the total number of a, i.e., worse than normal,
scores on part B of the DALDA questionnaire for both PRO
and CON. Notable increases in the number of fatigue-related
symptoms including, need for a rest, muscle pains, between-
session recovery, general weakness, sleep, and irritability
were prevalent after INT compared with NOR in both trials.
Increased dietary protein intake led to a very likely (97%)
attenuation (17; T11 AUC of ‘‘a’’ scores part B, DALDA for
INT+ REC) of increased symptoms of stress after a block of
high-intensity training.
GMS scores: POMS-65 questionnaire. The sub-
scales of the POMS-65 are presented in Figure 4. Iceberg
profiles were flattened, indicating increased stress, after INT
in both PRO and CON. Most notable changes after INT
were observed in fatigue and vigor. More reported feelings
BASIC SCIENCES
of fatigue were reported after INT compared with NOR in
both PRO (NOR = 7 T 2; INT = 15 T 1) and CON (NOR =
5 T 1; INT = 17 T 1). In contrast, feelings of vigor declined
after INT compared with NOR in both PRO (NOR = 16 T 7;
INT = 12 T 5) and CON (NOR = 16 T 3; INT = 8 T 2).
However, no clear effect of diet on feelings of fatigue, vigor,
depression, anger, and confusion after INT or REC were
observed compared with NOR. Likewise, GMS after INT
(PRO = 138 T 30; CON = 144 T 29) and REC (PRO = 114 T
36; CON = 104 T 25) compared with NOR (PRO = 119 T
36; CON = 110 T 31) were not clearly affected by additional
protein intake.
Psychometric ratings/subjective complaints. Av-
erage weekly fatigue ratings (Likert scale) were higher after
INT (5 T 1) and REC (4 T G1) compared with NOR (3 T G1)
in CON. A similar pattern was observed for PRO (NOR =
3 T 1; INT = 6 T 2; REC = 4 T 2). No clear effect of diet on
Medicine & Science in Sports & Exercised 603
 FIGURE 4—Iceberg profile (POMS-65) questionnaire after normal (NOR), intensified (INT), and recovery training (REC) in high-protein (PRO; A)
and normal protein (CON; B) conditions. Cyclists completed the POMS-65 questionnaire on waking in a rested state on a daily basis. Raw values are
expressed as means T SD.
fatigue, stress (data not shown), or sleep quality (data not
BASIC SCIENCES
shown) was observed after a block of high-intensity training.
DISCUSSION
This study was designed as a proof-of-concept investiga-
tion to examine the effect of additional protein intake on the
change in endurance performance after an intensified period
of training. When protein intake was similar to both the
habitual intake of recruited cyclists and recently published
recommended dietary protein requirements for endurance
athletes (1.2–1.4 gIkgj1 BMIdj1) (35), like those in previ-
ous studies (2,12,14,20), a substantial decrement in time trial
performance and mood state was observed immediately after
a block of high-intensity training. Our findings are novel in
providing preliminary evidence, suggesting that an increased
dietary protein intake of 3 gIkgj1 BMIdj1 attenuates psy-
chological symptoms of stress and may ameliorate decre-
ments in endurance performance after an intensified period
of training.
The practical relevance of the attenuated mean decrement
in time trial performance after INT when additional dietary
protein was administered was examined by an inferential
evaluation of our data set using confidence interval-based
statistics (17,18). Rather than declaring the value of the
statistic based on the probability of a null outcome, use of
the analysis of the likelihood of meaningful change has re-
cently been advocated to be a more intuitive and practical
statistical approach based directly on uncertainty in the true
value of the statistic (4,18). This approach determined that
intake of additional protein provides a È30% (possible)
chance of a beneficial effect and G1% chance of a harmful
effect on time trial performance directly after a block of
high-intensity training. This inference may be explained by
the È100-s (È5%) average attenuation of the decrement in
time trial performance designed to last È45 min in PRO
versus CON after INT compared with NOR. Furthermore,
the probability (48%) that attempts to restore endurance
performance with 1 wk of recovery training benefited from
an increased dietary protein intake existed. Thus, our data
604 Official Journal of the American College of Sports Medicine
Copyright © 2011 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
provide promising, albeit preliminary, evidence supporting
the suggestion that additional protein intake may attenuate
decrements in time trial performance after a block of high-
intensity training.
It must be acknowledged that our experimental design
may have contributed to the somewhat uncertain primary
experimental outcome, i.e., the probabilistic term assigned to
qualify the effect of additional protein intake on endurance
performance only reached possibly beneficial. Practical and
logistical considerations precluded tight scientific experimen-
tal control. Training load prescribed during the experimental
block was not supervised within a laboratory situation; rather,
autonomy was given to cyclists to select their own workload
within an HR range. Moreover, washout periods between
trials were not standardized (range = 2–4 wk). Hence, the
possibility exists that these factors combined contributed to
an increased variability in physiological responses to die-
tary intervention and subsequent compromise in reliability
of the time trial to assay endurance performance. A coeffi-
cient of variation G5% has been calculated for the determi-
nation of endurance performance in a glycogen-depleted
state using the time trial protocol used in the present study
when preceded by a normal training load (7). However,
equivalent reliability data do not exist within an intensified
training context. In addition, the intensified training model
used in the present study has been well established to reduce
performance immediately after the intense training period
(2,14). Hence, the unexpected observation that one subject
improved performance after INT compared with NOR in
PRO, exacerbated by a small sample size, may have further
reduced the chance of detecting a substantial effect of protein.
Thus, it may be argued that our hypothesis that additional
protein intake will result in the better maintenance of endur-
ance performance after a high-intensity block of training can
neither be comprehensively accepted nor refuted. However,
from a purely practical perspective, informing an athlete or
coach that a 30% possibly beneficial and G1% most unlikely
harmful effect of increasing dietary protein intake on endur-
ance performance during a high-intensity block of training
exists may be argued to be attractive.
http://www.acsm-msse.org
 It should be noted that these results may be applicable
only in the context of a relatively low CHO availability. The
cyclists in this study consumed a CHO intake equivalent to
6 gIkgj1 BMIdj1 in both trials. This level of CHO intake is
at the low end of the recommendation for endurance training
and well below that recommended for intense endurance
training (6). Thus, increased protein per se may not have
been the most salient factor. Previous research has focused
on the effect of CHO rather than protein nutrition for re-
ducing the negative effect of intense training. Similar to our
results, CHO feeding was shown to alleviate decrements in
endurance performance after intensified periods of training
by È10% (2,14). Depleted muscle glycogen concentrations
provide a potential explanation for impaired endurance
performance typically experienced after intensified training
(34). Because cyclists in the present study consumed a
moderate-CHO diet (6 gIkgj1 BMIdj1, i.e., lower than
intakes recommended (8–10 gIkgj1 BMIdj1) for intense
endurance training (6)), it is likely that muscle glycogen
stores were depleted (34), thus contributing to the amelio-
ration of performance decrements in our subjects.
At this suboptimal CHO intake, the increased dietary
protein intake may have indirectly increased CHO avail-
ability or muscle glycogen stores via gluconeogenic path-
ways. Support for this notion is exemplified by a study that
demonstrated that the rate of gluconeogenesis was elevated
by È40% with a high-protein diet, consequently accounting
for a large proportion of endogenous glucose availability
(23). Our urea data may provide indirect evidence that glu-
coneogenesis was increased during PRO. Urea concen-
trations were greater with increased protein intake after
intensified training, suggesting that more amino acids were
deaminated, possibly increasing the availability of carbon
skeletons for gluconeogenesis and glycogen synthesis or
oxidation to provide energy. In the present study, calculation
of glycogen oxidation or the determination of muscle gly-
cogen content could not be made. However, it is possible
that the better maintenance of endurance performance after
intensified training observed in the high-protein condition
may be attributed to a restored rate of CHO oxidation, par-
ticularly because CHO intake was suboptimal. Thus, the
high-protein intake simply may have partially countered
an inadequate CHO availability, rather than having a direct
effect per se.
A modulated endocrine response has been proposed as a
possible mechanism underpinning the better maintenance of
endurance performance when nutrition is manipulated dur-
ing intensified periods of training (14). The cumulative
effect of releasing high concentrations of stress hormones
during consecutive bouts of intense exercise has been sug-
gested to desensitize the hypothalamic–pituitary–adrenal
axis (16) and ultimately reduce availability of selected hor-
mones (36). This notion is supported by an attenuated peak
cortisol response to maximal exercise that we and others
(14,37) observed after intensified training. Furthermore, the
beneficial effect of CHO nutrition for the attenuation of
PROTEIN, INTENSE TRAINING, AND PERFORMANCE
Copyright © 2011 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
decrements in performance associated with intensified
periods of training has also previously been attributed to a
modulated endocrine response (2,14). CHO supplementa-
tion before, during, and after training was suggested to
attenuate the desensitization of the hypothalamic–pituitary–
adrenal axis after intensified training and was thus deemed
responsible for restoring the blunted peak cortisol response
(14). However, peak stress hormone concentrations mea-
sured in the present study were not different between con-
ditions. In addition, peak testosterone concentrations remained
within reported ‘‘normal’’ ranges (È3–10 ngImLj1) for
healthy males (3), irrespective of dietary protein intake. Thus,
whereas it is clear that CHO affects the endocrine response
to intensified training, the mechanism responsible for the
potentially better maintenance of endurance performance
with protein feeding does not seem to be related to a modu-
lated stress hormone or testosterone response.
BASIC SCIENCES
In addition to endocrine parameters, protein nutrition
failed to affect physiological and subjective markers of
increased training stress. In accordance with previous find-
ings, the number and severity of postexercise physiological
markers of stress, such as increased resting and sleeping HR
(9), fatigue, and stress ratings, were elevated after consecu-
tive days of excessive training (13). Increasing dietary pro-
tein intake during and after intensified training had no effect
on these physiological perturbations. Although it cannot be
completely ruled out, our data suggest that any improvement
in performance with protein feeding is unrelated to physio-
logical or biochemical markers of fatigue or a modulated
endocrine response.
The most clear and least speculative explanation respon-
sible for the possibly beneficial effect of additional protein
intake on the maintenance of endurance performance during
a block of high-intensity training may be related to indirect,
centrally driven mechanisms. In agreement with previous
findings (2,14,29), psychological symptoms of stress, in
particular the number of fatigue-related symptoms of stress
(i.e., ‘‘need for a rest’’ and ‘‘general weakness’’), deterio-
rated after overload training. CHO feeding has previously
been reported (2,14) to attenuate the negative psychological
symptoms of stress associated with intensified training. In
the present study, this athlete-specific DALDA question-
naire revealed that the number of psychological symptoms
of stress reported by cyclists during and after a period of
intensified training was attenuated when dietary protein in-
take was increased. The improved psychological status of
athletes during and after a short-term period of intensified
training in the high-protein trial potentially contributed to
the better maintenance of endurance performance after over-
load training. Previous research suggests that tyrosine sup-
plementation attenuates decrements in stress-related mood
(28); thus, it is possible that an increased availability of amino
acids, in particular tyrosine, mediated an improved mood
state in the protein condition. The utility of the clinically
based POMS questionnaire to monitor the mood state of
athletes should be interpreted with caution when applied to
Medicine & Science in Sports & Exercised 605
 an athletic population (29) and therefore may explain why
the beneficial effect of protein feeding on mood state was
not further substantiated using the POMS. Further research
should be conducted to fully elucidate the mechanism(s)
responsible for this beneficial effect of protein feeding on
psychological status.
Practical implications. In the context of an intensified
period of training, caution should prevail before replicating
dietary manipulations administered in the present study.
CHO remains the most important fuel to maximize endur-
ance performance during both normal and intensified periods
of training. Our experimental design was implemented to
maximize the chance of detecting an effect of protein feed-
ing; thus, energy balance during INT was achieved pre-
dominantly by increasing protein intake. During strenuous
endurance endeavors such as the Tour de France, whereby
daily energy expenditures consistently exceed 5000 kcal,
BASIC SCIENCES
protein intakes are regularly reported to be as high as
3 gIkgj1 BMIdj1 (32). However, typically, these athletes
concurrently increase CHO intake. In the present study,
irrespective of training stress, a standardized moderate-CHO
diet (6 gIkgj1 BMIdj1) was consumed, an intake previously
demonstrated to be insufficient for maintaining endurance
performance during intensified periods of training (2). A
recent study (24) demonstrated that, when cyclists maintained
normal weekly training volumes, a high-CHO diet rather than
an isoenergetic high-protein, moderate-CHO diet resulted in
improved endurance performance. Dietary CHO and protein
in that study were remarkably similar to that of our subjects
during PRO, further supporting the notion that the perfor-
mance of our subjects may have been influenced by a
somewhat inadequate CHO intake. Hence, we acknowledge
that the apparent beneficial effect of additional protein intake
for the better maintenance of endurance performance was
achieved under conditions whereby the CHO intake of
cyclists may be deemed suboptimal to support such a short-
term period of overload training. Moreover, a recent study
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this work was received from DSM Food Specialties, Delft, The
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There are no conflicts of interest for any of the authors.
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