Am J Physiol Endocrinol Metab 316: E741–E748, 2019.
First published February 19, 2019; doi:10.1152/ajpendo.00174.2018.
RESEARCH ARTICLE
Indicator amino acid oxidation protein requirement estimate in endurance-
trained men 24 h postexercise exceeds both the EAR and current athlete
guidelines
Arash Bandegan,1 Glenda Courtney-Martin,2,3,4 Mahroukh Rafii,2 Paul B. Pencharz,2,4,5
and Peter W. R. Lemon1
1
Exercise Nutrition Research Laboratory, School of Kinesiology, Western University, London, Ontario, Canada; 2Research
Institute, Hospital for Sick Children, Toronto, Ontario, Canada; 3Faculty of Kinesiology & Physical Education, University of
Toronto, Toronto, Ontario, Canada; 4Department of Nutritional Sciences, University of Toronto, Toronto, Ontario, Canada;
and 5Department of Paediatrics and Nutrition Science, University of Toronto, Toronto, Ontario, Canada
Submitted 4 May 2018; accepted in final form 14 February 2019
Bandegan A, Courtney-Martin G, Rafii M, Pencharz PB,
Lemon PW. Indicator amino acid oxidation protein requirement
estimate in endurance-trained men 24 h postexercise exceeds both the
EAR and current athlete guidelines. Am J Physiol Endocrinol Metab
316: E741–E748, 2019. First published February 19, 2019; doi:
10.1152/ajpendo.00174.2018.—Despite studies indicating increased
protein requirements in strength-trained or endurance-trained (ET)
individuals, the Institute of Medicine has concluded that “no addi-
tional dietary protein is suggested for healthy adults undertaking
resistance or endurance exercise,” and the controversy regarding
exercise effects on protein requirements continues. The objective of
this study was to determine the dietary protein requirement of healthy
young ET men (ⱖ1 yr training experience) 24 h post exercise (to
minimize any acute effects of the previous training session) by
measuring the oxidation of ingested L-[1-13C]phenylalanine to 13CO2
in response to graded intakes of protein (indicator amino acid oxida-
tion technique). Eight men [maximal oxygen consumption 64.1
ml·kg⫺1·min⫺1 (SD 3.7)] were each studied 24 h postexercise repeat-
edly with protein intakes ranging from 0.3 to 3.5 g·kg⫺1·day⫺1.
Protein was fed as an amino acid mixture based on the protein pattern
in egg, except for phenylalanine and tyrosine, which were maintained
at constant amounts across all protein intakes. For 2 days before the
study day, all participants consumed 1.6 g protein·kg⫺1·day⫺1. The
estimated average requirement (EAR) for protein was determined by
applying a nonlinear mixed-effects change-point regression analysis
to F13CO2 (label tracer oxidation in 13CO2 breath), which identified a
breakpoint in the F13CO2 in response to the graded amounts of
protein. The EAR for protein and the upper 95% confidence interval
were 2.1 and 2.6 g·kg⫺1·day⫺1, respectively. These data suggest that
the protein EAR for ET men 24 h postexercise exceeds the Institute of
Medicine EAR and established athlete guidelines by ~3.5- and 1.3-
fold, respectively.
athletes; endurance exercise; nitrogen balance; protein requirement;
stable isotope
INTRODUCTION
The current recommended dietary allowance (RDA) for
protein is based on the nitrogen balance (NB) technique, which
Address for reprint requests and other correspondence: P. Lemon, 2212
3M Centre, Western Univ., London, ON, Canada N6A3K7 (e-mail: plemon
@uwo.ca).
http://www.ajpendo.org 0193-1849/19 Copyright © 2019 the American Physiological Society
defines estimated average requirement (EAR) as the minimum
intake necessary to maintain nitrogen equilibrium (22). The
RDA exceeds the EAR [EAR ⫹ 2 standard deviations (SD)] to
ensure requirements are met for those whose needs exceed the
average (22). Interestingly, there are several reports from NB
studies on endurance exercise-trained (ET) individuals indicat-
ing protein intake recommendations between 1.2 and 1.6 g·kg
body mass⫺1·day⫺1 (14, 20, 28, 46, 61), yet the Institute of
Medicine (22) does not recognize these data. As a result, the
debate regarding protein needs of the physically active contin-
ues.
Although a classic tool that has provided significant data in
the past, NB measurements have several inherent problems
(38, 43). This is evident in the NB data used for protein require-
ment determination in ET individuals (14, 36, 61), in which
increasing dietary protein intakes result typically in linear un-
realistically positive NB. Equally concerning is an accommo-
dative NB response at low protein intakes via an increased
nitrogen reutilization, which produces an underestimation of
protein requirement (68). Also, NB at lower protein intakes
may be achieved through a compromise in some physiologi-
cally relevant processes, such as lesser enzyme activity upregu-
lation, decreased capillarization, and/or reduced mitochondrial
biogenesis with ET (60). All of this would be considered as
undesirable outcomes for ET. Furthermore and perhaps criti-
cally, application of NB for protein requirement determination
is unsuitable with exercise training (45) because these individ-
uals aim to consume optimal amounts of dietary protein to
induce a positive net protein status (anabolism) via maximal
protein synthesis rates over protein breakdown (catabolism) for
the best whole body and muscle remodeling/adaptation. Fi-
nally, skeletal muscle constitutes only ~30% of whole body
protein metabolism, and human protein need is not exclusive to
skeletal muscle but rather encompasses all body proteins and
associated processes. Therefore, the optimal protein require-
ment methodology for ET individuals needs to determine an
intake that maximizes whole body protein status (protein syn-
thesis minus protein breakdown) to repair/remodel all body
proteins necessary for the greatest training-induced adaptive
response. One such method is the indicator amino acid oxida-
tion (IAAO) technique, which is based on the partitioning of
E741
E742 PROTEIN REQUIREMENT IN ENDURANCE-TRAINED MEN
amino acids (AA) between whole body protein synthesis or
oxidation. Specifically, when dietary protein is inadequate, the
oxidation of all AA, including the indicator AA, will be
substantial. With increasing dietary protein, oxidation of the
indicator AA must decrease because more AA are being
incorporated into body protein. Once the dietary requirement is
met, the oxidation of the indicator AA plateaus, and the
resulting inflection or “breakpoint” is thought to be the mean
requirement or EAR (43).
To date, there are a few reports on protein EAR in ET using
the IAAO (2, 24, 42, 67), but, to our knowledge, chronic, i.e.,
with any acute effects of the previous training session mini-
mized, protein EAR of ET individuals using IAAO has not yet
been reported. Therefore, the purpose of this study was to
quantify the daily EAR for protein of ET men 24 h postexercise
using the IAAO technique. The 24-h postexercise time point
for measurement is critical because protein metabolism is
altered acutely postexercise.
METHODS
Potential candidates were invited for an interview via postings at
the Western University campus as well as at community athletic and
recreation centers. Study details were reviewed, and any who decided
to participate signed an informed consent statement previously ap-
proved by Western University’s Health Sciences Research Ethics
Board. The inclusion criteria were 1) healthy men between 18 and 40
yr of age; 2) ⱖ1 yr of endurance-training experience, ⱖ6 days/wk,
~1–1.5 h/day; 3) body mass stable (⬍4-kg mass gain or loss within the
past 6 mo); 4) nonsmoker; 5) ⬍8 g alcohol/day; 6) non-medication
user; and 7) without allergies to milk or milk products (because Boost
meal replacement and whey protein isolate were given as part of the
experimental diet). All participants passed a physical activity readi-
ness questionnaire (PAR-Q) health survey (63) and performed a
maximal oxygen consumption (V̇O2max) test on a motorized treadmill.
Briefly, after a 5-min warm-up on the treadmill (Desmo Pro, Wood-
way USA, Waukesha, WI), breath-by-breath gas analysis (Vmax
Legacy, SensorMedics, Yorba Linda, CA) was completed during a
progressive incremental-slope test (starting at 0% grade and speed of
11–12 km/h) to determine V̇O2max. The treadmill slope was increased
by 2% every min until volitional exhaustion (test duration varied from
6 to 12 min). Oxygen uptake and heart rate (using a Polar RST200TM
heart rate monitor, Polar Electro Inc., Lachine, QC, Canada) were
measured continuously. V̇O2max was taken as the greatest value
averaged over a 30-s collection period. All the participants achieved
a plateau in oxygen consumption and attained a heart rate within 10
beats/min of their age-predicted maximum. Although the ET athletes
participated in endurance training primarily, they also completed 1–2
days/wk of mobility, flexibility, and strength training (whole body
circuit strength training).
Study design. The minimally invasive (oral infusion) IAAO tech-
nique was used (6, 50) to determine daily dietary protein requirement
of the ET athletes measured 24 h postexercise to minimize any acute
effects of the previous training session. During the initial laboratory
visit, which followed a 12-h overnight fast, both resting energy
expenditure (REE) and body composition [fat-free mass (FFM) and
fat mass] of each athlete were measured using open-circuit indirect
calorimetry (Vmax Legacy), and air displacement plethysmography
with a BodPod (Life Measurements, Concord, CA), respectively.
Each dietary protein quantity given was studied over a 3-day period
(two adaptation days followed by an IAAO study day). We have
demonstrated previously that adaptation to changing intakes, unlike
NB, takes hours rather than days (40). During the adaptation days,
participants received Boost (Nestlé) meal replacement as a mainte-
nance diet supplemented with Polycose (Abbot Nutrition) and lactose-
free, gluten-free whey protein isolate (Kaizen Protein) providing 1.6 g
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00174.2018 • www.ajpendo.org
protein·kg⫺1·day⫺1 and 1.7 ⫻ REE energy. To maintain their sched-
uled training routine, ET participants continued to train on the two
adaptation days. The activities on adaptation days were based on each
participant’s noncompeting-season routine, generally a moderate-
intensity activity such as a run or bike ride (1–1.5 h/day) or any
combination of flexibility, mobility, stretching work, or swimming
skill correction clinic. These activities were duplicated before every
study day. To ensure adequate energy intake during adaptation days,
3-day dietary records of participants were analyzed and compared
with our calculated energy requirement based on REE and physical
activity level guidelines (22). On the third day (the study day),
participants arrived in the morning after a 12-h fast and were assigned
randomly to receive test protein intakes ranging from 0.3 to 3.5
g·kg⫺1·day⫺1 with 1.5 ⫻ REE as total energy intake. Three and two
participants were each tested at 4 and 6 protein intakes, and one
participant at each of 3, 7, and 9 protein intakes for a total of 43 IAAO
studies. Each participant was studied 24 h following their previous
training session, and all 3-day study periods were separated by at least
1 wk.
Study diets. The adaptation and study diets provided all of the
participant’s macronutrient needs on the basis of the current guide-
lines of the Academy of Nutrition and Dietetics, Dietitians of Canada,
and the American College of Sport Medicine (62). Macronutrient
breakdown of the diet on adaptation days was carbohydrate [6.7
g·kg⫺1·day⫺1 (SD 0.6)], protein [1.6 g·kg⫺1·day⫺1 (SD 0.1)], and fat
[0.7 g·kg⫺1·day⫺1 (SD 0.1)]. During the two adaptation days, the
daily diet was consumed as four equal meals, and participants did not
consume any other food items except one cup of clear tea or coffee
and water ad libitum. Participants were also provided with a multivi-
tamin supplement (Centrum, Wyeth Consumer Health Care) and a
stool softener/laxative (RestoraLax, Bayer) daily for the duration of
all studies.
On the third study day (IAAO day), the participants arrived at the
laboratory in the morning following an overnight fast and consumed
eight hourly isoenergetic meals, each meal representing one-twelfth of
his daily energy requirement. The study day diet consisted of a
protein- and AA-free powder (PDF1, Mead Johnson), flavored drink
crystals (Tang and Kool-Aid, Kraft Foods), grape-seed oil, a crystal-
line AA mixture (Ajinomoto Amino Science LLC) patterned after egg
protein (Table 1), and protein-free cookies. Energy intake was set at
1.5 ⫻ REE, and the carbohydrate content of the diets was adjusted
based on the protein to keep the diets isoenergetic. Macronutrient
breakdown for carbohydrate and fat for the study day diets was 5.0
(SD 0.9) and 1.2 g·kg⫺1·day⫺1 (SD 0.2), respectively.
Tracer protocol. An oral priming doses of 0.176 mg NaH13CO3/kg
body mass (99 atom percent excess, Cambridge Isotope Laboratories)
and 0.66 mg L-[1-13C]phenylalanine (Phe)/kg started with the fifth
hourly meal. In addition, a dose of L-[1-13C]Phe (1.2 mg·kg⫺1·h⫺1)
was started with the fifth meal and continued hourly for the remaining
4 h of the study. The quantity of L-[1-13C]Phe supplied during the last
4 h of the study was subtracted from the diet to provide a total intake
of 25 mg Phe·kg⫺1·day⫺1, and tyrosine was provided at 40
mg·kg⫺1·day⫺1 to ensure an excess of tyrosine (56).
Sample collection and analysis. During each study day, three
baseline breath and urine samples were collected at 45, 30, and 15 min
before the tracer protocol began, and five total breath and urine
samples were collected, one every 30 min beginning at 2.5 h after
administration of the tracer (L-[1-13C]Phe), to establish that an isoto-
pic steady state was attained. Breath samples were collected in
disposable Exetainer tubes (Labco) with a collection mechanism
(Easy-Sampler, Quintron) that permitted the removal of dead-space
air. Breath samples were stored at room temperature, and urine
samples were stored at ⫺20°C until analysis. During each study day,
the rate of carbon dioxide production (V̇CO2) was measured immedi-
ately after the fifth meal for a period of 20 min with an indirect
calorimeter (Vmax Legacy). Expired 13CO2 enrichment was measured
with a continuous-flow isotope ratio mass spectrometer (CF-IRMS
 PROTEIN REQUIREMENT IN ENDURANCE-TRAINED MEN E743
Table 1. Amino acid composition of reference protein and test protein intakes
Test Protein Intake, g/kg

Reference Proteina , mg/g 0.3 1.0 1.5 2.0 2.5 3.0 3.5

L-Alanine 61.5 18.45 61.5 92.3 123 153 184 215

L-Arginine-HCLb 75.1 22.53 75.1 113 150 187 225 263
L-Asparagine 33.3 9.99 33.3 50.0 66.6 83 99.9 116
L-Aspartic acid 33.3 9.99 33.3 50.0 66.6 83.2 99.9 116
L-Cysteine 22.1 6.63 22.1 33.2 44.2 55.2 66.3 77.3
L-Glutamine 56.6 16.98 56.6 84.9 113 141 169 198
L-Glutamic acid 56.6 16.98 56.6 84.9 113 141 169 198
Glycine 33.3 9.99 33.3 50.0 66.6 83.2 99.9 116
L-Histidine 22.7 6.81 22.7 34.1 45.4 56.7 68.1 79.4
L-Isoleucine 62.8 18.84 62.8 94.2 126 157 188 219
L-Leucine 83.3 24.99 83.3 125 167 208 250 291
L-Lysine-HCLb 75.7 22.71 75.7 114 151 189 227 265
L-Methionine 29.6 8.88 29.6 44.4 59.2 74.0 88.8 103
L-Phenylalaninec 30.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0
L-Proline 41.9 12.57 41.9 62.9 83.8 104 125 146
L-Serine 83.9 25.17 83.9 126 168 209 251 293
L-Threonine 47.1 14.13 47.1 70.7 94.2 117 141 165
L-Tryptophan 15.6 4.68 15.6 23.4 31.2 39.0 47 54.6
L-Tyrosined 40.0 40.0 40.0 40.0 40.0 40.0 40.0 40.0
L-Valine 70.3 21.09 70.3 105 141 175 211 246
Participants received several test protein quantities ranging from 0.3 to 3.5 g·kg⫺1·day⫺1. aRepresents the egg protein composition. bActual amounts of amino
acids were as follows: arginine 62.1 mg/g and lysine 60.6 mg/g. cL-Phenylalanine intake was kept constant at 25.0 mg·kg⫺1·day⫺1. dL-Tyrosine intake was kept
constant at 40.0 mg·kg⫺1·day⫺1.

20/20 isotope analyzer, PDZ Europa). Enrichments were expressed as
atom percent excess compared with a reference standard of com-
pressed CO2. Urinary L-[1-13C]Phe enrichment was analyzed by an
API 4000 triple quadrupole mass spectrometer (Applied Biosystems/
MDS Sciex) in positive electrospray ionization mode (50). Isotopic
enrichment was expressed as mole percent excess and calculated from
peak area ratios at isotopic steady state both at baseline and plateau.
Estimation of isotope kinetics. L-[1-13C]Phe kinetics was calculated
using the stochastic models described previously (33). Isotopic steady
state in the tracer enrichment at baseline and plateau was represented
as unchanging values of L-[1-13C]Phe in urine and 13CO2 in breath.
Steady-state Phe flux (␮mol·kg⫺1·h⫺1) was calculated from the dilu-
tion of the orally administered L-[1-13C]Phe into the metabolic pool
by using the enrichment of L-[1-13C]Phe in urine (6). The rate of
appearance of 13CO2 in breath (F13CO2 ␮mol·kg⫺1·h⫺1), after the
oxidation of ingested L-[1-13C]Phe, Phe oxidation (␮mol·kg⫺1·h⫺1),
and net Phe status (␮mol·kg⫺1·h⫺1) were calculated according to the
model of Matthews et al. (33) using a factor of 0.82 to account for
carbon dioxide retained in the body’s bicarbonate pool (19).
Statistical analysis. All results are reported as means (SD). Statis-
tical analyses were performed by using SAS (Version 9.2.1; SAS
Institute Inc.). Significance was set at P ⬍ 0.05. Participants were
assigned randomly to differing protein intakes with protein quantity as
the independent variable. The effect of protein intake on Phe flux was
tested using a mixed linear model (PROC MIXED) with subject as a
random variable. The EAR for protein was determined by applying a
nonlinear mixed-effects model (PROC NLMIXED; SAS Institute) to
the F13CO2, Phe oxidation and net Phe status data (2). Observations
within subjects were regarded as statistically dependent. Confidence
intervals (CI) were obtained by following the standard asymptotic
theory of the maximal likelihood estimation. The model minimizing
the Akaike information criterion was regarded as the model with the
best fit (18). The following statistical model was used, accounting for
correlations within observations from the same subject:
Yid ⫽ ␤0 ⫹ bi ⫹ ␤11共xid ⬎ xcp兲共xid – xcp兲 ⫹ ␧id ,
where Yid ⫽ F13CO2 or Phe oxidation at the dose of the protein of i,
xid is the dose amount of the test protein intake of the i-th participant,
εid are random errors that are independently normally distributed with
a mean of 0 and variance of ␴2, ␤0 is the left line intercept, bi is the
random intercept that incorporates within-subject correlation, ␤11 is
the left line slope, xcp is the breakpoint, and the slope for xid is 0 for
xid more than breakpoint. Comparisons between the results of the two
studies presented in Table 4 were made by using a t-test with P ⬍
0.05.
RESULTS
Participant characteristics. Eight ET men (ⱖ1 yr of consis-
tent endurance-training experience, ⱖ6 days/wk, ~1–1.5 h/day
with minimal strength training in their routine, ~1–2 days/wk)
participated in the study during their noncompeting season. Of
the eight participants recruited, there were one, one, three, and
three triathlete, long-distance runner, cross-country runners,
and duathletes (cycling and running), respectively, with a
V̇O2max of 64.1 ml·kg⫺1·min⫺1 (SD 3.7) (Table 2). Their
habitual protein intake based on 3-day dietary record analysis
was 2.1 g·kg⫺1·day⫺1 (SD 0.9), which is ~2.6 times the current
RDA.
Phe kinetics. Phe flux was not affected within each ET
individual by the different protein intakes (P ⫽ 0.14) as
required by the IAAO method (Table 3). This provides
evidence that the precursor pool for the IAAO did not
change significantly with increasing test protein intakes and
suggests that the changes in oxidation were inversely pro-
portional to whole body protein synthesis. Whole body Phe
flux based on body mass and FFM was 67.4 (SD 3.7) and
74.5 ␮mol·kg⫺1·h⫺1 (SD 3.8), respectively.
Nonlinear mixed-effects change-point regression analysis of
the F13CO2, Phe oxidation, and net Phe status data resulted in
a breakpoint at a protein intake of 2.1 g·kg⫺1·day⫺1
(r2 ⫽ 0.68), i.e., the rate of 13CO2 released from the oxidation
of L-[1-13C]Phe (F13CO2) (Fig. 1) and Phe oxidation (Fig. 2)
declined in response to increasing protein intakes up to 2.1
g·kg⫺1·day⫺1, such that additional increases in protein intake

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00174.2018 • www.ajpendo.org

E744 PROTEIN REQUIREMENT IN ENDURANCE-TRAINED MEN

Table 2. Physical characteristics and daily energy and (increased protein synthesis) eventually reaching a breakpoint,
macronutrient intake of the endurance-trained men studied i.e., the EAR. Importantly, our data follow a consistent trend to
those reported in previous IAAO-derived protein EAR for
Characteristics Value
sedentary individuals and our recent IAAO measures for body-
Age, y 26.6 (5.8) builders, all of which exceed the published NB protein require-
Mass, kg 68.2 (2.0) ments set by the Institute of Medicine (2, 21, 22, 50). Appar-
Height, m 1.8 (0.1)
Body fata, % 9.8 (5.0) ently, there are limitations inherent in the NB method, includ-
FFM, kg 61.2 (6.0) ing overestimation of intake and underestimation of losses,
V̇O2max, ml·kg⫺1·min⫺1 64.1 (3.7) slow physiological urea pool adaptation to altered protein
REEb, kcal·kg⫺1·day⫺1 24.9 (3.1) intake, confounding effects of energy intake, and true nitrogen
Energy intakec, kcal·kg⫺1·day⫺1 40.7 (3.9) accretion below the limits of detection that are responsible for
Carbohydrate intakec, g·kg⫺1·day⫺1 5.1 (0.3)
Protein intakec, g·kg⫺1·day⫺1 2.1 (0.9) these differences, as mentioned earlier (38). Furthermore, as
Fat intakec, g·kg⫺1·day⫺1 1.3 (0.3) protein intake increases from deficient to excess, the efficiency
of its utilization decreases, hence the physiological response is
All values are means (SD), n ⫽ 8. FFM, fat-free mass; REE, resting energy
expenditure; V̇O2max, maximum oxygen consumption. aBody fat was measured nonlinear. Therefore, the common application of linear regres-
using Bod Pod (Life Measurements). bResting energy expenditure measured by sion analysis to these data leads to an underestimation of the
open-circuit indirect calorimetry (Vmax Legacy, SensorMedics). cEnergy true requirement. In support of this point, when repeated
intake and macronutrient breakdown were based on a 3-day dietary record measures of NB data with different protein intakes on the same
analysis.
participants were analyzed using a biphase linear regression
model, the protein EAR by NB or IAAO was similar (21).
did not result in changes in F13CO2 or Phe oxidation values. Consequently, NB versus IAAO discrepancies in the literature
This indicates that the incorporation of the Phe for protein may be largely due to differences in the regression model used.
synthesis plateaued at a protein intake of 2.1 g·kg⫺1·day⫺1 and Finally, NB requires an extensive adaptation period (7–10
suggests that this is the protein EAR of these ET individuals. days) to any diet manipulation (38), so assessing chronic
The upper 95% CI of the breakpoint was at 2.6 g·kg⫺1·day⫺1, protein EAR 24 h postexercise in ET athletes via NB would be
approximating the RDA for protein of ET individuals. The nearly impossible.
lower CI was 1.6 g·kg⫺1·day⫺1. Our IAAO results with ET athletes reveal a greater EAR
In addition, nonlinear mixed-effects change-point regression versus our earlier IAAO study on sedentary young men (21) by
analysis of calculated net Phe status (synthesis minus break- ~2.2 times; Table 4. As the methodology was identical for both
down) was negative, with lower protein intakes showing a of these studies, this comparison is consistent with the conclu-
linear increase as the protein intake quantities increased from sion that endurance training increases dietary protein needs
0.3 to 3.5 g·kg⫺1·day⫺1 (r2 ⫽ 0.65), with breakpoint at 2.1 (27, 62). Whole body Phe flux at rest for our ET men was
g·kg⫺1·day⫺1 and a lower and upper CI of 1.6 and 2.6 15.2% greater than these sedentary young men (P ⬍ 0.05) (21)
g·kg⫺1·day⫺1, respectively (Fig. 3). but when expressed relative to FFM, this difference was
reduced to a nonsignificant 4%. During the two prestudy days,
DISCUSSION
we provided a protein intake of 1.6 g·kg⫺1·day⫺1 based on the
guidelines of the Academy of Nutrition and Dietetics, Dieti-
Typically, the protein intake of ET athletes [1.8 g·kg⫺1· tians of Canada, and the American College of Sport Medicine
day⫺1 (SD 0.4)] exceeds the current RDA (60). Similarly, the (62) for endurance athletes from previous NB data (28, 60).
protein intake [2.1 g·kg⫺1·day⫺1 (SD 0.9)] of our ET men was This was less than the habitual protein intake of our partici-
greater than the RDA. Furthermore, previous studies assessing pants [2.1 g·kg⫺1·day⫺1 (SD 0.9)], so it might be concluded
protein requirements for ET individuals utilizing the classic that this difference influenced our measures, but, importantly,
NB technique have reported values from 0.94 to 1.6 g·kg⫺1· this is not the case because, unlike NB measures, habitual
day⫺1 (17.4%–100% ⬎RDA) (14, 20, 36, 46, 61). Our IAAO
data 2.1 g·kg⫺1·day⫺1 (Fig. 1) extend the evidence that dietary
protein requirements are greater for ET individuals because our Table 3. Protein intakes and phenylalanine flux in
requirement estimate is at least 31% ([2.1⫺1.6]/1.6 ⫻ 100) endurance-trained men
greater than the previous ET NB data and 250% ([2.1⫺
Test Protein Intakes, Phenylalanine Flux,
0.6]/0.6 ⫻ 100) greater than the current protein EAR (14, 22, Subject g·kg⫺1·day⫺1 ␮mol·kg⫺1·h⫺1
28, 29, 60, 61). Moreover, several variables affect exercise
1 0.5, 0.9, 1.2, 1.7, 2.1, 2.4 56.5 (1.7)
estimates including varying training experience, intensity, du- 2 0.9, 1.3, 1.5, 1.9, 2.3, 2.8, 3.5 63.6 (1.6)
ration, volume of training, and nutritional status, as well as 3 0.3, 1.1, 1.1, 1.6, 1.6, 2.3, 1.9, 2.9, 3.3 63.1 (1.3)
measurement technique limitations (27, 38, 39), and the debate 4 0.8, 1.0, 1.5, 1.9 64.1 (2.1)
about exact protein needs for the chronically physically active 5 0.6, 1.0, 1.4, 1.7, 2.0, 2.5 60.8 (2.0)
continues. 6 0.8, 1.3, 1.7 67.9 (2.4)
7 1.1, 1.8, 2.4, 3.0 65.2 (2.0)
With the model we used, oxidation of L-[1-13C]Phe (indica- 8 0.5, 1.2, 1.7, 2.0 64.0 (1.7)
tor) at several protein intakes was measured as a reverse proxy All 65.6 (1.8)
for whole body protein synthesis, a relationship that has been
Values are means (SD). No significant differences in phenylalanine flux
confirmed previously (51). Consequently, as dietary protein were observed within each subject across all test protein intakes (0.3–3.5
intake increases, the rate of L-[1-13C]Phe oxidation decreases g·kg⫺1·day⫺1; P ⫽ 0.14). Each participant completed a minimum of three test
because of a greater nonoxidative disposal of L-[1-13C] Phe intakes for a total of 43 studies.

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00174.2018 • www.ajpendo.org

 PROTEIN REQUIREMENT IN ENDURANCE-TRAINED MEN
Fig. 1. Protein intake and 13CO2 production from phenylalanine oxidation
(F13CO2) in young endurance-trained men (43 experiments). Individual values
for each athlete are represented by different symbols. The breakpoint repre-
sents the estimated average requirement (EAR) for protein. A mixed-effects
change-point regression analysis identified a breakpoint and upper 95% CI for
the relation between protein intake and phenylalanine oxidation to be 2.1 and
2.6 g·kg⫺1·day⫺1 (r2 ⫽ 0.68), respectively. d, day.
protein intakes do not alter protein or AA requirements as
determined with the IAAO technique (12, 13, 40, 49).
Interestingly, a recent IAAO study conducted immediately
after an endurance exercise bout (24) reported a protein EAR
of 1.65 g·kg⫺1·day⫺1 [~2.75-fold greater than current EAR
(22)], which is consistent with the idea that endurance exercise
increases the protein requirements. However, this estimate is
only 79% of our EAR (2.1 g·kg⫺1·day⫺1) measured 24 h
postendurance exercise, and we suspect the different postexer-
cise measurement time points are largely responsible for this
discrepancy. For example, in the fed state, increases in protein
synthesis occur by 1–2 h poststrength exercise and remain
elevated for ~24 h. Thereafter, protein synthesis decreases
gradually but can be elevated even 48 h postexercise (7, 32,
47). Unfortunately, the postexercise time course with endur-
ance exercise is not as well established, but there are reports
indicating a mixed muscle protein synthesis increase (17%–
22%) for as long as 48 h postexercise with 4 wk to 4 mo of
endurance training, respectively (48, 57). In contrast, using
muscle biopsy samples at 4 h postendurance exercise, investi-
gators could not find any elevated mitochondrial protein syn-
thetic response to protein ingestion (5, 8). Perhaps 4 h posten-
durance exercise is too soon to detect any increases in protein
synthesis because of the delayed posttranslational response of
mitochondrial protein synthesis (5, 11). Indeed, in the fed state
following dynamic exercise (one leg kicking at 67% of Wmax),
the fractional protein synthetic rate for sarcoplasmic, myofi-
brillar, and tendon collagen protein in quadriceps muscle were
all increased by 6 h, peaking at 24 h postexercise (37).
Consequently, acute effects of the previous exercise bout linger
into recovery so that a differing protein EAR based on imme-
diate postendurance-exercise measures versus 24 h post should
not be surprising. Of course, training experience is likely
another important factor (27), and this may have differed
between these studies. Furthermore, it is well established that
low muscle glycogen availability increases protein require-
ments because of increased protein degradation (4, 29), and
this likely differed between these two studies as well, even if
the exercise glycogen use was similar, because postexercise
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00174.2018 • www.ajpendo.org
E745
carbohydrate intake was nearly double in the Kato et al. study
(24) versus ours. Consequently, an increase in circulating
insulin resulting from this greater carbohydrate ingestion could
reduce protein degradation (3, 16), Phe flux, and the protein
EAR. Apparently based on a 17% greater Phe flux in their
endurance athletes [68.8 ␮mol·kg⫺1·h⫺1 (SD 8.5)] versus
IAAO measures in nonexercising individuals [58.5 ␮mol·kg⫺1·
h⫺1 (SD 14.0)] (21), Kato et al. (24) assumed that any reduced
protein breakdown due to this substantial carbohydrate intake
was minimal. However, as mentioned, reduced protein degra-
dation postexercise would reduce the protein EAR relative to
that measured 24 h postexercise. Together, these postexercise
changes in protein synthesis and degradation over 24 h postex-
ercise could explain the lower protein EAR in the Kato et al.
study (24). A full understanding of how acute endurance
exercise affects the protein EAR must await a time-course
study.
Relative to the increased protein EAR versus sedentary
individuals (21), there are a number of acute and chronic
physiological effects of exercise that could increase the protein
EAR for ET athletes. These include an increased AA oxidation
(alanine, asparagine, aspartate, glutamate, lysine) (26, 58),
especially for the branched chain AA (leucine, isoleucine and
valine) during exercise (25, 46, 54), as well as greater mito-
chondrial biogenesis (17), upregulated repair and remodeling
processes in the muscles used over time (e.g., increased aerobic
enzymes, capillaries, hemoglobin, and myoglobin) (48, 57),
altered immune response (40, 41, 64), increased oxidative
stress (23), and/or increased protein synthesis in important
support tissues, e.g., connective/soft tissues and bone (1, 37).
For example, exercise oxidation of AA can contribute up to
~5%–10% of the total energy requirement during exercise
depending on exercise duration, intensity, and carbohydrate
availability (25, 29, 53), which would need to be compensated
for postexercise. In addition, exercise promotes conversion of
AA to glucose following exercise via gluconeogenic pathways,
and as a result, the rate of urea production is stimulated (52).
Fig. 2. Protein intake and phenylalanine (Phe) oxidation in young endurance-
trained men (43 experiments). Individual values for each athlete are repre-
sented by different symbols. The breakpoint represents the estimated average
requirement (EAR) for protein. A mixed-effects change-point regression anal-
ysis identified a breakpoint (EAR) and upper 95% CI for the relation between
protein intake and Phe oxidation to be 2.1 and 2.6 g·kg⫺1·day⫺1 (r2 ⫽ 0.68),
respectively. d, day.
E746 PROTEIN REQUIREMENT IN ENDURANCE-TRAINED MEN
Fig. 3. Relationship between protein intake and net phenylalanine (Phe) status
in young endurance-trained men (43 experiments). Individual values for each
athlete are represented by different symbols. The breakpoint represents the
estimated average requirement (EAR) for protein. A mixed-effects change-
point regression analysis identified a breakpoint (EAR) and upper 95% CI for
the relation between protein intake and net Phe status to be 2.1 and 2.6
g·kg⫺1·day⫺1 (r2 ⫽ 0.65), respectively. d, day.
Following the cessation of muscle contraction is when rates
of muscle protein synthesis have the potential to be accelerated
beyond the rates of proteolysis, allowing protein status to
become positive (10, 52). However, without postexercise pro-
tein intake, protein synthesis must be limited because of a
decreased availability of AA. For example, provision of 10 –16 g
of protein after a bout of moderate endurance exercise (60%– 65%
V̇O2max for ~60 min) enhanced muscle protein synthesis and net
muscle protein status despite a negative whole body protein
status (30, 31). In our study, provision of dietary protein at
lower quantities (from 0.3 to 1.3 g·kg⫺1·day⫺1) resulted in a
negative Phe status, meaning that Phe breakdown (proteolysis)
was greater than its nonoxidative disposal (protein synthesis),
presumably because of a limited AA supply (Fig. 3). It was at
1.3 g·kg⫺1·day⫺1 where Phe status approached zero (Fig. 3).
Moreover, a neutral (zero) status may not be conducive to an
optimal exercise training-induced adaptation, as it relates only
to the protein intake needed to recover losses. Relative to the
dietary protein requirement for ET athletes, the optimal protein
intake would be the protein quantity that results in adaptation
rather than accommodation (45), where accommodation in-
volves increased nitrogen utilization efficiency and lower
whole body protein synthesis (68), which occurs when there is
insufficient protein intake (61). For an ET athlete, the ideal
adaptation response would correspond to the greatest net whole
body protein status to cover all relevant repair and remodeling
processes involved. As such, maximal whole body Phe status,
i.e., a plateau in whole body protein synthesis (nonoxidative
Phe disposal) relative to breakdown, would likely promote
optimal muscle adaptation. This status was reached in our ET
participants at a protein intake of 2.1 g·kg⫺1·day⫺1 (Table 4).
Interestingly, this EAR is similar to the habitual protein intake
of our ET athletes (Table 2) and represents ~20% of their total
daily energy intake measured during their noncompeting sea-
son. However, to cover those individuals with greater than
average needs, an EAR of 2.1 g·kg⫺1·day⫺1 equates to an RDA
of ~2.6 g·kg⫺1·day⫺1 (Table 4), which is ~3.2-fold greater than
current protein RDA. Regardless, this intake is well within the
acceptable macronutrient intake range (10%–35% of total daily
energy intake) as defined by the Institute of Medicine (22).
Nonetheless, reporting percent macronutrient intake is limited
because absolute intakes depend on energy intakes, which can
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00174.2018 • www.ajpendo.org
differ widely. Our perspective is that g/kg body mass recom-
mendations are best. Finally, care should to be taken when
using measures of muscle protein synthesis to assess dietary
protein need because clearly changes in muscle protein syn-
thesis do not necessarily reflect whole body protein metabolism
(9, 10, 57). Consequently, we believe that measurement of net
postexercise whole body protein status is the technique of
choice for protein requirement determinations, as it reflects the
integration of all relevant physiological/remodeling processes
(both anabolic and catabolic) for better training-induced adap-
tations, including but not limited to those in muscle tissue. For
example, with endurance exercise, damage and repair of
splanchnic tissues (e.g., hypoxia-mediated small intestinal in-
jury) consequent to the shunting of blood away from the gut to
exercising muscles can be substantial (65). Symptoms such as
nausea, vomiting, abdominal angina, and bloody diarrhea are
experienced by up to 70% of ET athletes (44). In addition,
following protein ingestion, significant quantities of AA are
retained in the splanchnic area functioning as a labile pool of
indispensable AA (59). In fact, first-pass splanchnic extraction
of AA accounts for 20%–90% of ingested AA (34, 35).
Furthermore, splanchnic tissue has a high protein turnover rate
and, therefore, is capable of retaining AA, then later releasing
those AA when needed by other tissues (e.g., by muscle during
exercise or sleep) (9). Moreover, splanchnic protein breakdown
is increased during exercise (66), and it is possible that both
splanchnic extraction of AA as well as protein synthesis is
enhanced postexercise, as replacement (55).
In summary, our data suggest that ET men have a protein
EAR that exceeds both the current EAR and earlier require-
ment estimates for athletes based on the NB technique, al-
though timing of the measures relative to the previous exercise
bout is likely very important. As both NB and IAAO method-
ologies have their critics (2, 15, 38, 39, 43, 50), future longi-
tudinal studies with treatment groups assigned differing protein
intake quantities in combination with exercise performance
data are needed to confirm these data. Unfortunately, in addi-
Table 4. Comparison between young endurance-trained men
(current study) and young healthy sedentary men who
participated in another protein EAR study conducted with
IAAO method
Variable Young Endurance-Trained Mena Sedentary Young Menb
Age, y 26.6 (5.8) 26.8 (2.0)
N 8 8
Height, m 1.8 (0.1) 1.7 (2.6)
Mass, kg 68.2 (2.0) 69.6 (3.7)
FFM, kg 61.2 (6.0) 56.4 (5.4)
Phenylalanine flux,
␮mol·kg⫺1 BM·h⫺1 67.4* (3.7) 58.5 (14.0)
␮mol·kg⫺1 FFM·h⫺1 74.5 (3.8) 71.9 (18.7)
Protein EAR,
g·kg⫺1 BM·day⫺1 2.1 0.93
g·kg⫺1 FFM·day⫺1 2.3 1.1
Protein RDAc,
g·kg⫺1 BM·day⫺1 2.6 1.2
g·kg⫺1 FFM·day⫺1 2.9 1.5
Values are means (SD), comparisons were performed by t-test. BM, body
mass; EAR, estimated average requirement; FFM, fat-free mass; IAAO,
indicator amino acid oxidation; RDA, Recommended Dietary Allowance.
a
Current study. bData from (21). cBased on upper 95% CI of breakpoint.
*Different from sedentary, P ⬍ 0.05.
 PROTEIN REQUIREMENT IN ENDURANCE-TRAINED MEN
tion to being labor intensive and expensive, such studies are
very difficult because of the number of variables [genetic
capacity, gender, training intensity and duration, state of train-
ing, energy balance, carbohydrate intake, and type of protein
ingested (60)] that must be controlled. Perhaps other novel
protein requirement methodologies are needed to quantify
precisely how exercise alters protein need. Regardless, it has
been suggested that meeting dietary protein need is unlikely to
be a concern for ET individuals, assuming an adequate energy
intake; however, this conclusion was based on NB data (44,
60), which are at least 30% less than our IAAO EAR measure
of 2.1 g·kg⫺1·day⫺1. Finally, together with our earlier protein
EAR measures in bodybuilders (2), these ET IAAO data
suggest that the dietary protein intake recommendation set by
the Institute of Medicine are inadequate for those involved in
regular exercise training.
ACKNOWLEDGMENTS
A. Bandegan gratefully acknowledges Dr. Daniel Moore (University of
Toronto) for his critique of the original data set.
This trial was registered at clinicaltrials.gov as NCT02621294.
GRANTS
This work was supported in part by Mead Johnson Nutritionals, Abbott
Nutrition, and Nestlé Health Science.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
A.B. and P.W.L. conceived and designed research; A.B. performed exper-
iments; A.B., G.C.-M., M.R., and P.W.L. analyzed data; A.B. and P.W.L.
interpreted results of experiments; A.B. prepared figures; A.B. drafted manu-
script; A.B., G.C.-M., P.B.P., and P.W.L. edited and revised manuscript; A.B.,
G.C.-M., M.R., P.B.P., and P.W.L. approved final version of manuscript.
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