J Appl Physiol 106: 1394–1402, 2009.

First published November 26, 2008; doi:10.1152/japplphysiol.90333.2008.

HIGHLIGHTED TOPIC Regulation of Protein Metabolism in Exercise and Recovery

Coingestion of protein with carbohydrate during recovery from endurance

exercise stimulates skeletal muscle protein synthesis in humans
Krista R. Howarth, Natalie A. Moreau, Stuart M. Phillips, and Martin J. Gibala
Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada
Submitted 27 February 2008; accepted in final form 21 November 2008

Howarth KR, Moreau NA, Phillips SM, Gibala MJ. Coinges-
tion of protein with carbohydrate during recovery from endurance
exercise stimulates skeletal muscle protein synthesis in humans.
J Appl Physiol 106: 1394 –1402, 2009. First published November
26, 2008; doi:10.1152/japplphysiol.90333.2008.—Coingestion of pro-
tein with carbohydrate (CHO) during recovery from exercise can affect
muscle glycogen synthesis, particularly if CHO intake is suboptimal.
Another potential benefit of protein feeding is an increased synthesis rate
of muscle proteins, as is well documented after resistance exercise. In
contrast, the effect of nutrient manipulation on muscle protein kinetics
after aerobic exercise remains largely unexplored. We tested the hypoth-
esis that ingesting protein with CHO after a standardized 2-h bout of
cycle exercise would increase mixed muscle fractional synthetic rate
(FSR) and whole body net protein balance (WBNB) vs. trials matched for
total CHO or total energy intake. We also examined whether postexercise
glycogen synthesis could be enhanced by adding protein or additional
CHO to a feeding protocol that provided 1.2 g CHO䡠kg⫺1 䡠h⫺1, which is
the rate generally recommended to maximize this process. Six active men
ingested drinks during the first 3 h of recovery that provided either 1.2 g
CHO䡠kg⫺1 䡠h⫺1 (L-CHO), 1.2 g CHO ⫹ 0.4 g protein䡠kg⫺1 䡠h⫺1 (PRO-
CHO), or 1.6 g CHO䡠kg⫺1 䡠h⫺1 (H-CHO) in random order. Based on a
primed constant infusion of L-[ring-2H5]phenylalanine, analysis of biopsies
(vastus lateralis) obtained at 0 and 4 h of recovery showed that muscle
FSR was higher (P ⬍ 0.05) in PRO-CHO (0.09 ⫾ 0.01%/h) vs. both
L-CHO (0.07 ⫾ 0.01%/h) and H-CHO (0.06 ⫾ 0.01%/h). WBNB
assessed using [1-13C]leucine was positive only during PRO-CHO,
and this was mainly attributable to a reduced rate of protein break-
down. Glycogen synthesis rate was not different between trials. We
conclude that ingesting protein with CHO during recovery from
aerobic exercise increased muscle FSR and improved WBNB, compared
with feeding strategies that provided CHO only and were matched for
total CHO or total energy intake. However, adding protein or additional
CHO to a feeding strategy that provided 1.2 g CHO䡠kg⫺1 䡠h⫺1 did not
further enhance glycogen resynthesis during recovery.
protein turnover; stable isotopes; amino acids; glycogen
NUTRIENT INGESTION during recovery can profoundly alter the acute
metabolic response to exercise and potentially augment training-
induced adaptations in skeletal muscle (8). A classic example by
Bergstrom and colleagues (3) showed that carbohydrate (CHO)
ingestion stimulated skeletal muscle glycogen synthesis during
recovery from prolonged exercise. The general mechanisms re-
sponsible for this phenomenon have been well established (10),
but questions remain regarding the optimal type, timing, and
amount of CHO necessary to maximize glycogen synthesis and
the effect of ingesting other macronutrients (6).
Coingestion of protein with CHO can augment glycogen
synthesis during recovery, particularly if CHO intake is sub-
optimal (9, 28, 33), but most evidence suggests that feeding
CHO at a rate ⱖ 1.2 g䡠kg⫺1 䡠h⫺1 negates the benefit of added
protein (6, 10, 11, 27). Irrespective of the potential to modulate
glycogen synthesis, protein ingestion during recovery may
confer other benefits, including the synthesis and repair of muscle
proteins involved in energy metabolism and force production
(23). Numerous studies have shown that protein ingestion,
alone or with CHO, stimulates mixed skeletal muscle protein
synthesis after acute resistance-based exercise (17, 22, 24, 26,
31). However, the effect of nutrient manipulation on muscle
protein kinetics during recovery from endurance exercise re-
mains largely unexplored. Levenhagen et al. (14) reported that
muscle protein synthesis was increased after aerobic exercise
when protein was added to carbohydrate-fat supplement com-
pared with the supplement without protein. However, the authors
could not conclusively discern whether the addition of protein was
responsible for the increased protein synthetic response or
whether the effects were related to an increase in energy intake per
se. In addition, the study (14) relied on arteriovenous measure-
ments, as opposed to a direct measurement of muscle protein
synthesis, which can be obtained by combining stable isotope
infusion with muscle biopsy sampling (32).
The primary purpose of the present study was to determine
whether ingesting protein with CHO during recovery from
prolonged exercise would increase mixed skeletal muscle pro-
tein fractional synthetic rate (FSR) and improve whole body
protein balance compared with CHO alone. A secondary pur-
pose was to determine whether adding protein or additional
CHO to a feeding strategy that provided 1.2 g CHO䡠kg⫺1 䡠h⫺1,
which is the rate generally recommended to maximize postex-
ercise glycogen synthesis (6, 11), would further augment this
process during recovery. On three occasions during recovery
from a 2-h bout of standardized cycle exercise, subjects in-
gested drinks that provided either 1.2 g CHO䡠kg body
wt⫺1 䡠h⫺1, 1.2 g CHO ⫹ 0.4 g protein䡠kg⫺1 䡠h⫺1, or 1.6 g
CHO䡠kg⫺1 䡠h⫺1. Our primary hypothesis was that coingestion
of protein with CHO would increase FSR and whole body
protein balance compared with drinks matched for total CHO
or total energy intake. We also tested the secondary hypothesis
that ingesting protein or additional CHO would result in a
higher rate of muscle glycogen synthesis compared with the
control trial that provided 1.2 g CHO䡠kg⫺1 䡠h⫺1.

Address for reprint requests and other correspondence: M. J. Gibala, Dept. The costs of publication of this article were defrayed in part by the payment
of Kinesiology, McMaster Univ., Hamilton, Ontario, Canada L8S 4K1 (e-mail: of page charges. The article must therefore be hereby marked “advertisement”
gibalam@mcmaster.ca). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE
METHODS
Subjects
Six healthy men (age 22 ⫾ 1 yr; mass 90 ⫾ 5 kg; height 184 ⫾ 2
cm; body mass index 26.4 ⫾ 0.8 kg/m2) volunteered for the study.
The subjects were recreationally active and habitually engaged in a
variety of activities that included running, cycling, weightlifting, and
intramural sports several times per week, but none were specifically
training for a particular sport or event. Their peak oxygen uptake
(V̇O2 peak), determined using an incremental test on an electronically
braked cycle ergometer (Lode BV, Excalibur Sport V2.0) and an
on-line gas collection system (Moxus Modular VO2 System, AEI
Technologies, Pittsburgh, PA), was 4.4 ⫾ 0.3 l/min. A preliminary
screening process was employed to confirm that subjects were free of
risk factors associated with cardiovascular, pulmonary, or metabolic
disease. The experimental procedures and potential risks were fully
explained to the subjects before the study, and all subjects provided
written, informed consent. The experimental protocol was approved by
the Hamilton Health Sciences/Faculty of Health Sciences, McMaster
University Research Ethics Board.
Overview of Experimental Design
Each subject served as his own control and performed three
experimental trials in random order, separated by at least 7 days. Each
trial consisted of a 2-h standardized bout of exercise (see below)
followed by a 4-h recovery period during which subjects ingested one
of three experimental beverages (Fig. 1). All trials were performed in
an identical manner, the only difference being the composition of the
beverage ingested during recovery. Beverages were ingested at a rate
of 750 ml/h, in 15-min intervals for the first 3 h of recovery, and
formulated to deliver either 1.2 g CHO 䡠 kg body mass⫺1 䡠 h⫺1 (L-
CHO), 1.2 g CHO ⫹ 0.4 g protein 䡠 kg⫺1 䡠 h⫺1 (PRO-CHO; matched to
L-CHO in terms of total CHO ingested), or 1.6 g CHO 䡠 kg⫺1 䡠 h⫺1
(H-CHO; matched to PRO-CHO in terms of total energy ingested).
The CHO source was maltodextrin (Glucidex IT 19, Roquette Frère),
and the protein source was a hydrolyzed whey protein concentrate
(American Casein, HLA-198). To make the drinks comparable in
taste, 5 g of Sucralose (Splenda), 2.5 g of sodium chloride, and orange
powder flavoring were added to 750 ml of each beverage. To maintain
constant infusion of L-[ring-2H5]Phe, an amount of isotope equivalent
to 9% of the Phe present in the whey protein was added to the
PRO-CHO drinks. Beverages were ingested during the first 3 h of
recovery since pilot testing revealed some individuals experienced
considerable gastrointestinal distress (particularly bloating and an
urge to vomit) if drinks were ingested after this time (i.e., over the
entire 4-h recovery period).
Physical Activity and Nutritional Controls
Subjects were asked to keep their habitual exercise pattern and
dietary intake as constant as possible over the course of the experi-
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1395
ment, but given the within-subject design (i.e., each subject served as
his own control), these parameters were not standardized between
subjects. Subjects were specifically instructed to perform no physical
activity, aside from activities of daily living, for 48 h before each trial.
Subjects were also asked to maintain food records during the 48 h
before each trial. Following the first experimental trial, subjects were
instructed to replicate their individual nutritional pattern over the
course of the second and third trials and again record food intake,
noting any deviations from the first trial. Food records were subse-
quently analyzed (Nutritionist Five dietary analysis software, First
Data Bank, San Bruno, CA), and results confirmed no difference in
total energy intake or macronutrient composition between trials (data
not shown). In addition to these general nutrition controls, all subjects
were provided with a standardized, prepackaged meal on the day
before each experimental trial. Subjects were instructed to ingest the
meal as breakfast at ⬃0700 on the day of the trial, after having fasted
overnight. The meal provided 700 kcal and was derived from 82%
carbohydrate, 10% fat, 8% protein. Subjects were instructed not to
consume any other food or drink, except for water, before reporting to
the laboratory.
Experimental Trial Details
Subjects arrived at the laboratory at ⬃0900 on the day of each
experimental trial. On arrival, subjects were weighed and a catheter
was inserted into an antecubital vein. After a resting blood sample was
obtained, a baseline breath sample was collected into a 100-liter
Douglas bag that was connected to an on-line gas collection system
(Moxus) for the determination of carbon dioxide output (V̇CO2). A
10-ml sample of expired air was transferred into a Vacutainer tube and
used for subsequent analysis of background breath 13CO2 enrichment
using isotope ratio mass spectrometry (IRMS) as previously described
(29). Subjects then received a bolus infusion of NaH13CO3 (0.295
mg/kg) to prime the bicarbonate pool and a primed constant infusion
of two stable isotopically labeled amino acids, L-[ring-2H5]phenylalanine
(prime, 2 ␮mol/kg; infusion, 0.05 ␮mol䡠kg⫺1 䡠min⫺1) and [1-13C]leucine
(prime, 1 mg/kg; infusion, 1 mg䡠kg⫺1 䡠h⫺1) for the determination of whole
body and skeletal muscle protein kinetics as described below.
At ⬃1000, or ⬃3 h following ingestion of the standardized meal,
subjects mounted an ergometer (Lode) and initiated a 2-h bout of
standardized variable-intensity cycling to reduce muscle glycogen
content. The protocol was modeled after Kuipers et al. (13) and
consisted of 12 ⫻ 10-min stages that alternated between 50% V̇O2 peak
(174 ⫾ 7 W) and a higher workload that began at 80% V̇O2 peak
(278 ⫾ 11 W) and decreased by 5% V̇O2 peak every other stage to a
final workload of 55% V̇O2 peak (191 ⫾ 8 W). Immediately following
exercise, the lateral portion of one thigh was prepared for the extrac-
tion of a needle biopsy sample from the vastus lateralis muscle (2) and
a second catheter was inserted into the contralateral arm for blood
sampling. A biopsy sample and blood sample were obtained within 10
min of the cessation of exercise (which corresponded to ⬃5 h
Fig. 1. Overview of experimental protocol.
106 • APRIL 2009 • www.jap.org
1396 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE
following the standardized breakfast), and these samples were con-
sidered “0 h” samples that corresponded to the start of the recovery
period. Blood samples were collected at 15 min intervals for the initial
hour of recovery and every 30 min thereafter, and a second biopsy
sample was obtained after 4 h of recovery. All biopsies for a given
trial were obtained from separate incision sites on the same leg, and
legs were alternated between trials. Expired breath samples were
collected in triplicate during the final 15 min of the first and fourth
hours of recovery.
Gastrointestinal Distress Measurements
Subjects were asked to complete a gastrointestinal (GI) distress
questionnaire immediately after exercise and each hour of recovery.
The questionnaire was adapted from Jentjens et al. (11) and assessed
11 parameters of GI distress including nausea, bloating, GI cramps,
need to belch, need to vomit, diarrhea, urge to urinate, urge to
defecate, headache, dizziness, and body chills. Each parameter was
ranked on a scale from 1 to 10, with 1 being absence of any distress
and 10 being distress strongly present (minimum score 11, maximum
score 110).
Muscle Analyses
Upon removal from the leg, each muscle sample was immediately
frozen in liquid nitrogen and subsequently stored at ⫺86°C before
analyses. Muscle samples were subsequently divided into two pieces
while still frozen. One portion was freeze-dried, powdered, and
subsequently dissected free of obvious blood and connective tissue.
After this process was completed, the freeze-dried samples were
stored at ⫺86°C before analysis of glycogen using flourometry and
amino acids using HPLC. The remaining piece of frozen muscle was
used for subsequent determination of mixed muscle fractional syn-
thetic rates (FSR) using gas chromatography mass spectrometry
(GCMS).
Glycogen. A ⬃2-mg aliquot of freeze-dried muscle was incubated
in 2.0 N HCl and heated for 2 h at 100°C to hydrolyze the glycogen
to glucosyl units. The solution was subsequently neutralized with an
equal volume of 2.0 N NaOH and analyzed for glucose using an
enzymatic assay adapted for fluorometry (20).
Amino acids. Freeze-dried muscle was extracted on ice using 0.5 M
perchloric acid (PCA) (containing 1 mM EDTA), neutralized with 2.2
M KHCO3, and the resulting supernatant was used for the determi-
nation of free amino acids using HPLC. The concentrations of amino
acids were determined using a protocol described by Moore et al. (18).
Briefly, extracts were derivatized before injection using Waters
AccQ 䡠 Fluor reagent kit (Millford, MA) by heating for 30 min at 55°C
to form the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate de-
rivative of all physiological amino acids. Samples and standards
(Sigma, St. Louis, MO) were run on a Waters 2695 HPLC separations
module (Millford, MA) through a Nova-Pak C18, 4-␮m column to
separate the amino acids. The amino acids were detected using a
Waters 474 scanning fluorescence detector (Millford, MA) with ex-
citation and emission wavelengths of 250 nm and 395 nm, respec-
tively. Amino acid peak areas were integrated, compared with known
standards, and analyzed using the Waters Millenium 32 software
package. The method achieved reliable separation of 17 of the 20
physiological amino acids.
Mixed muscle FSR. Muscle samples collected at 0 h and 4 h were
used for the measurement of mixed muscle FSR based on L-[ring-
2
H5]phenylalanine infusion as described by Wilkinson et al. (29).
Briefly, frozen muscle samples were weighed and ice-cold acetonitrile
(100 ␮l/mg) was added to extract the intracellular free amino acids.
Samples were then manually homogenized, vortexed for 10 min, and
centrifuged at 15,000 rpm for 2 min at 4°C. The supernatant was
collected and the procedure repeated. The pooled supernatant was
then dried under N2 gas for later analysis of the muscle intracellular
free Phe enrichments. The remaining muscle pellets were washed
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twice with distilled H2O, once with absolute ethanol, and then freeze
dried overnight. The dried pellets were subsequently weighed and
hydrolyzed with 6 N HCl (400 ␮l/mg) for 24 h at 110°C. A 300-␮l
aliquot of each bound protein hydrolysate was passed over a C18
reverse-phase chromatography spin column (Harvard Apparatus, Hol-
liston, MA). The eluted samples were then dried under N2 gas.
Samples were derivatized to the tert-butyldimethyl silyl (tBDMS)
derivative of phenylalanine using 50 ␮l N-methyl-N-(tert-butyldim-
ethyl) trifluoroacetamide (MTBSTFA) ⫹ 1% tert-butyl-dimethylchlo-
rosilane (TBDMCS) (Pierce Chemical, Rockford, IL) ⫹ 50 ␮l anhy-
drous acetonitrile and heated for 15 min at 100°C. The Phe isotopic
enrichments were determined on an electron-impact ionization GC-MS
(GC: Agilent 6890N; MS: Hewlett-Packard 5973). Ions were selec-
tively monitored at mass-to-charge (m/z) ratios of 234 and 239 for Phe
enrichment in the intracellular muscle extracts, and 237 and 239 in the
bound muscle hydrolysate. Bound muscle protein enrichments were
determined using the standard curve method (21). FSR was calculated
using the equation published by Wolfe (32).
Blood Analyses
Blood samples were collected into heparinized and nonheparinized
tubes. One 200-␮l aliquot of heparinized whole blood was combined
with 1,000 ␮l of 0.6 N perchloric acid (PCA), vortexed, and centri-
fuged, and the supernatant was collected and stored at ⫺30°C. The
PCA extract was subsequently analyzed for glucose using an enzy-
matic assay adapted for fluorometry (20). A second 200-␮l aliquot of
heparinized whole blood was combined with 1,000 ␮l of 0.6 N PCA,
vortexed, centrifuged, and neutralized with 500 ␮l of 1.25 N KHCO3.
The supernatant was collected and stored at ⫺30°C for later analysis
of amino acids using HPLC as described above. The remaining
heparinized whole blood was centrifuged and the plasma was col-
lected and stored at ⫺30°C for subsequent analysis using GCMS. The
isotope enrichment of plasma ␣-ketoisocaproic acid (␣-KIC) was
measured on an electron-impact ionization GCMS (GC: Agilent
6890N; MS: Hewlett-Packard 5973, Palo Alto, CA) using a method
described previously (24, 29). Nonheparinized tubes were centrifuged
and serum was collected and stored at ⫺30°C for subsequent analysis
of insulin using a radioimmunoassay kit (RIA) (Coat-A-Count, Diag-
nostic Products, Los Angels, CA). Area under the curve (AUC) for
insulin was measured as total area over 4 h minus baseline.
Breath Analyses
The ratio of 13CO2 to 12CO2 was measured in breath samples using
an automated breath analysis system (BreathMat Plus, Thermo Finni-
gan, San Jose, CA) with a method we have described previously (29).
Values for breath samples measured in triplicate were corrected for
baseline breath 13CO2 enrichment and averaged.
Calculation of Whole Body Protein Kinetics
Calculations of whole body Leu flux (Q), oxidation (O), breakdown
(B), nonoxidative Leu disposal (NOLD), and net balance (NBAL) for
the 1-h and 4-h time points of recovery were made using the equations
described previously (16), with correction for ingested protein and an
estimated splanchnic extraction of 33% based on the results of Arnal
et al. (1). A bicarbonate retention factor (c) of 0.83 was used for the
retention of carbons in the blood bicarbonate pool during feeding (25).
Choice of Amino Acid Tracers
[1-13C]leucine was used for the determination of whole body
protein kinetics because this tracer allows measurement of kinetics
that primarily assess the impact of exercise on skeletal muscle rather
than other tissues. The measurement of whole body leucine kinetics
facilitates derivation of a number of parameters that are much harder
to obtain using other tracers due, for the most part, to the ease of
obtaining a representative precursor (i.e., ␣-KIC). Thus, in a scenario
106 • APRIL 2009 • www.jap.org
 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE 1397
in which muscle is active and a good tracer is available, leucine is an Table 1. Whole body leucine kinetic data
excellent choice as opposed to another tracer that may not show
sensitivity to changes. L-[ring-2H5]phenylalanine was used for the L-CHO H-CHO PRO-CHO
determination of muscle FSR since it is a highly deuterated and 1h 4h 1h 4h 1h 4h
“heavy” isotope (i.e., 5 atomic mass units greater than the endogenous
compound). Flux‡ 143⫾19 122⫾11 135⫾24 117⫾15 160⫾8† 207⫾8*
NOLD§ 96⫾11 79⫾9 104⫾22 80⫾13 111⫾8 67⫾22
Statistical Analyses Breakdown‡ 122⫾10 110⫾10 128⫾24 111⫾15 10⫾3* 37⫾8*

All muscle and blood data were analyzed using a two-factor
(treatment ⫻ time) repeated-measures ANOVA, except for muscle
FSR and glycogen synthesis rates, which were analyzed using a
one-factor (treatment) repeated-measures ANOVA. When a signifi-
cant main effect or interaction was identified, data were subsequently
analyzed using a Tukey honestly significant difference post hoc test.
Integrated AUC calculations were performed using GraphPad Prism
3.0 (GraphPad Software, San Diego, CA) and analyzed using a
one-factor (treatment) ANOVA. Significance for all analysis was set
at P ⱕ 0.05. All data are presented as means ⫾ standard error of the
mean (SE).
RESULTS
Isotope Analysis
Average blood plasma, muscle intracellular and breath iso-
tope enrichments are summarized in Fig. 2 and show subjects
achieved isotopic steady state.
Whole Body Leucine Kinetics
Leucine flux (Table 1) and oxidation (Fig. 3A) were higher
during PRO-CHO compared with H-CHO and L-CHO (main
effect treatment, P ⬍ 0.05), with both variables being higher at
4 h vs. 1 h (P ⬍ 0.05). NOLD was lower after 4 h vs. 1 h (main
effect time, P ⬍ 0.05), with no difference between trials (Table
1). Whole body protein breakdown was lower at 1 and 4 h of
recovery during PRO-CHO vs. the other trials (P ⬍ 0.05)
(Table 1). Leucine net balance was highest at 1 h of recovery
Values are means ⫾ SE; n ⫽ 6. NOLD, nonoxidative leucine disposal. L-CHO,
1.2 g carbohydrate (CHO)䡠kg⫺1 䡠h⫺1; H-CHO, 1.6 g CHO䡠kg⫺1 䡠h⫺1; PRO-CHO,
1.2 g CHO䡠kg⫺1 䡠h⫺1 ⫹ 0.4 g protein䡠kg⫺1 䡠h⫺1. *P ⬍ 0.05 vs. other treatments at
the same time point. †P ⬍ 0.05 vs. 4 h in the same trial. ‡PRO-CHO⫽L-CHO and
H-CHO (main effect treatment; P ⬍ 0.05). §Main effect for time (P ⬍ 0.05).
during the PRO-CHO trial (P ⬍ 0.05) (Fig. 3B). While leucine
net balance decreased from 1 to 4 h in all trials (main effect
time, P ⬍ 0.05), it remained positive in the PRO-CHO trial
whereas it was negative in the other two trials (main effect,
P ⬍ 0.05) (Fig. 3B).
Muscle FSR
Muscle FSR was higher (P ⬍ 0.05) during recovery in
PRO-CHO compared with both H-CHO and L-CHO (Fig. 4).
Muscle Glycogen
Muscle glycogen content was similar between trials after
exercise and increased to a similar extent in all trials over the
4-h period of recovery (main effect for time, P ⬍ 0.05) (Fig. 5).
There was no difference between trials in muscle glycogen
synthesis rate during recovery (L-CHO, 23 ⫾ 3; H-CHO, 25 ⫾
7; PRO-CHO, 25 ⫾ 4 mmol䡠kg dry wt⫺1 䡠h⫺1).
Muscle Amino Acids
Muscle amino acids are summarized in Table 2. Total
branched-chain amino acid (BCAA) content was higher (P ⬍

L-CHO
H-CHO
PRO-CHO

Fig. 2. Mean plasma (A and C), intracellular (B),

and breath (D) enrichments, expressed as tracer to
tracee (tr/T) ratios. Values are means ⫾ SE; n ⫽
6. Some error bars have been omitted for clarity.
␣-KIC, ␣-ketoisocaproic acid. L-CHO trial, 1.2 g
carbohydrate (CHO) 䡠 kg⫺1 䡠 h⫺1. H-CHO trial,
1.6 g CHO 䡠 kg⫺1 䡠 h⫺1. PRO-CHO, 1.2 g CHO ⫹
0.4 g protein 䡠 kg⫺1 䡠 h⫺1.

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1398 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE
Fig. 3. Whole body leucine oxidation (A) and net balance (B) during recovery
from prolonged exercise while ingesting either 1.2 g CHO 䡠 kg⫺1 䡠 h⫺1 (L-CHO),
1.6 g CHO 䡠 kg⫺1 䡠 h⫺1 (H-CHO), or 1.2 g CHO ⫹ 0.4 g protein 䡠 kg⫺1 䡠 h⫺1
(PRO-CHO). Values are means ⫾ SE; n ⫽ 6. *P ⬍ 0.05 vs. other treatments at
the same time point. †P ⬍ 0.05 vs. 4 h in the same trial. ‡Main effect for treatment,
P ⬍ 0.05. §Main effect for time, P ⬍ 0.05.
0.04) after 4 h of recovery during PRO-CHO vs. the other two
trials (Fig. 6A), and this was mainly due to changes in leucine
and isoleucine (Table 2). With respect to changes over time,
the content of aspartate, methionine, phenylalanine, proline,
and serine decreased from 1 to 4 h of recovery, with no
differences between trials (main effects, P ⬍ 0.05).
Blood Amino Acids
Blood amino acids are summarized in Table 3. After 1 h of
recovery, the concentrations of alanine, leucine, isoleucine,
valine, arginine, lysine, phenylalanine, proline, threonine, and
tyrosine were higher in PRO-CHO vs. the other trials and
remained higher for the remainder of the recovery (P ⬍ 0.05).
Total amino acid were higher during the PRO-CHO trial
Fig. 4. Mixed muscle protein fractional synthetic rate (FSR) during 4 h of recovery
from prolonged exercise while ingesting either 1.2 g CHO䡠kg⫺1 䡠h⫺1 (L-CHO), 1.6 g
CHO䡠kg⫺1 䡠h⫺1 (H-CHO), or 1.2 g CHO ⫹ 0.4 g protein䡠kg⫺1 䡠h⫺1 (PRO-CHO).
Values are means ⫾ SE; n ⫽ 6. *P ⬍ 0.05 vs. other treatments.
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Fig. 5. Muscle glycogen concentration over 4 h of recovery from prolonged exercise
while ingesting either 1.2 g CHO䡠kg⫺1 䡠h⫺1 (L-CHO), 1.6 g CHO䡠kg⫺1 䡠h⫺1 (H-
CHO), or 1.2 g CHO ⫹ 0.4 g protein䡠kg⫺1 䡠h⫺1 (PRO-CHO). Values are means ⫾
SE; n ⫽ 6. §Main effect for time, P ⬍ 0.05. dw, dry wt.
compared with the L-CHO trial by 1 h of recovery and were
higher than both CHO trials by 2.5 h of recovery (P ⬍ 0.05).
Total BCAA concentrations increased during the first hour of
recovery during the PRO-CHO trial compared with the CHO
alone trials and were significantly greater by 1 h of recovery
and remained higher throughout the remainder of the recovery
(P ⬍ 0.05) (Fig. 6B).
Blood Glucose and Serum Insulin
Blood glucose was higher after 60 –180 min of recovery vs.
immediately postexercise, but there was no difference between
treatments (main effect for time, P ⬍ 0.05; data not shown).
Serum insulin increased after 90 min and remained elevated for
the remainder of the recovery compared with immediately
postexercise, but there was no difference between trials (main
effect for time, P ⬍ 0.05; data no shown). The AUC for insulin
was not different between trials (L-CHO, 79 ⫾ 17; H-CHO,
90 ⫾ 22; PRO-CHO, 104 ⫾ 18 ␮IU䡠ml⫺1 䡠min⫺1).
Gastrointestinal Distress
Total GI distress scores were higher at 3 and 4 h of recovery
(reaching peak values of ⬃35) compared with immediately
postexercise (main effect for time, P ⬍ 0.05), but there was no
difference between trials.
DISCUSSION
The major finding from the present study was that coinges-
tion of protein with CHO during recovery from endurance
exercise increased mixed skeletal muscle FSR and induced a
more positive whole body net protein balance compared with
drinks matched for total CHO or total energy intake. This study
was unique in that it was the first to directly examine the effect
of manipulating CHO and protein ingestion during recovery
from endurance exercise on muscle FSR using the needle
biopsy technique. However, our data are consistent with the
work of Levenhagen et al. (14, 15), who used arterial-venous
difference measurements to show that adding protein to a
carbohydrate-fat supplement increased leg protein net balance
after exercise (1 h of cycling or recumbent cycling at 60%
V̇O2 peak) compared with the supplement without protein. How-
ever, those studies (14, 15) did not employ a trial matched for
total energy intake, and thus the authors could not conclusively
discern whether the increased muscle FSR was attributable to
106 • APRIL 2009 • www.jap.org
 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE 1399
Table 2. Muscle amino acids
L-CHO H-CHO PRO-CHO

0h 4h 0h 4h 0h 4h

Ala 7.0⫾0.7 7.7⫾1.0 7.2⫾0.5 7.3⫾0.5 7.2⫾1.1 7.1⫾0.2

Asn 1.0⫾0.1 0.9⫾0.2 1.1⫾0.1 0.8⫾0.1 1.1⫾0.1 1.0⫾0.1
Asp§ 1.3⫾0.3 0.5⫾0.1 1.8⫾0.4 0.6⫾0.1 1.1⫾0.3 0.6⫾0.1
Arg 27.0⫾3.2 26.1⫾2.6 27.1⫾1.9 23.0⫾2.3 27.5⫾4.2 22.4⫾2.2
Glu 4.9⫾0.2 4.5⫾0.7 7.0⫾0.9 5.7⫾0.9 5.5⫾0.7 5.5⫾0.9
Gln 29.7⫾1.5 30.5⫾2.3 34.3⫾2.7 33.5⫾3.1 32.3⫾4.1 31.1⫾2.8
Gly 6.4⫾0.5 6.4⫾1.0 6.6⫾0.6 6.2⫾0.7 6.8⫾0.8 5.9⫾0.4
His 1.6⫾0.0 1.7⫾0.3 1.5⫾0.2 1.7⫾0.1 1.7⫾0.1 1.6⫾0.1
Ile‡§ 0.4⫾0.0 0.2⫾0.0 0.4⫾0.0 0.3⫾0.0 0.4⫾0.0 0.5⫾0.0*
Leu‡§ 0.7⫾0.1 0.4⫾0.0 0.7⫾0.0 0.5⫾0.0 0.7⫾0.0 0.8⫾0.1*
Lys 2.1⫾0.2 1.8⫾0.2 2.4⫾0.3 1.8⫾0.2 2.3⫾0.4 2.5⫾0.4*
Met§ 0.3⫾0.0 0.2⫾0.0 0.3⫾0.0 0.2⫾0.0 0.3⫾0.0 0.2⫾0.0
Phe§ 0.3⫾0.0 0.2⫾0.0 0.3⫾0.0 0.3⫾0.0 0.3⫾0.0 0.3⫾0.0
Pro§ 5.0⫾0.6 4.3⫾0.4 4.3⫾0.4 3.9⫾0.3 5.2⫾0.5 4.7⫾0.4
Ser§ 1.4⫾0.1 1.1⫾0.2 1.7⫾0.3 1.1⫾0.1 1.6⫾0.2 1.2⫾0.2
Tau 29.7⫾2.8 26.2⫾2.0 31.0⫾3.8 27.8⫾3.1 30.9⫾5.4 26.1⫾5.4
Thr 3.7⫾0.6 2.5⫾0.8 4.3⫾0.7 3.3⫾0.6 3.9⫾0.7 4.6⫾0.3†
Tyr 0.4⫾0.0 0.2⫾0.0 0.4⫾0.0 0.3⫾0.0 0.4⫾0.0 0.3⫾0.0
Val§ 1.1⫾0.1 0.7⫾0.1 1.0⫾0.1 0.7⫾0.1 1.1⫾0.1 1.1⫾0.1
Total AA 124.1⫾7.7 116.2⫾8.9 133.4⫾10.0 118.9⫾10.0 130.3⫾15.0 117.6⫾12.1
Values are means ⫾ SE in mmol/kg dry wt; n ⫽ 6. *P ⬍ 0.05 vs. L-CHO and H-CHO at same time point. †P ⬍ 0.05 vs. L-CHO at same time point.
‡PRO-CHO ⬎ L-CHO and H-CHO (main effect treatment; P ⬍ 0.05). §Main effect for time (p ⬍ 0.05). AA, amino acids. See Table 1 for definitions of L-CHO,
H-CHO, and PRO-CHO trials.

the addition of protein per se or simply higher energy intake.
The present data confirm that protein ingestion was necessary
to stimulate protein synthesis, since muscle FSR was lower
during the H-CHO trial that was matched to PRO-CHO in
Fig. 6. Total branched-chain amino acid (BCAA) concentrations in muscle (A)
and blood (B) during 4 h of recovery from prolonged exercise while ingesting
either 1.2 g CHO 䡠 kg⫺1 䡠 h⫺1 (L-CHO), 1.6 g CHO 䡠 kg⫺1 䡠 h⫺1 (H-CHO), or 1.2 g
CHO ⫹ 0.4 g protein 䡠 kg⫺1 䡠 h⫺1 (PRO-CHO). Values are means ⫾ SE; n ⫽ 6.
*P ⬍ 0.05 vs. other treatments at the same time point. ‡Main effect for treatment
(PRO-CHO ⬎ L-CHO and H-CHO; P ⬍ 0.05). §Main effect for time, P ⬍ 0.05.
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terms of total energy intake. Our design could not resolve the
specific type of proteins that were responsible for the increase
in mixed muscle FSR. However, we recently found that 45 min
of cycling at 75% of V̇O2 peak elicited an increase in mitochon-
drial protein synthesis (30), and thus it is tempting to speculate
that the rise in mixed muscle protein synthesis we observed
with protein ingestion in our study was due at least in part to
increased mitochondrial protein synthesis.
An increased availability of blood and muscle amino acids,
especially the essential amino acids (EAA), has been associated
with an increased muscle FSR at rest and following resistance-
based exercise (22, 26). In the present study, there was an increase
in the concentration of the EAA (leucine, valine, isoleucine,
arginine, histidine, lysine, phenylalanine, threonine, and tyrosine)
in the blood in the PRO-CHO trial by 1 h of recovery that was
greater than that of the CHO-only trials, and the values remained
higher for the rest of the recovery period. While the present study
did not address potential mechanisms, the higher concentration of
EAAs would presumably ensure that their availability was not
limiting for the increased protein synthetic rate. Insulin has also
been shown to increase resting muscle protein synthesis when the
availability of amino acids is not limited (4), although the effect
following exercise is equivocal (5). Miller et al. (17) showed that
when glucose was added to an amino acid drink ingested during
recovery from resistance exercise, there seemed to be a synergistic
effect on muscle protein synthesis. In the present study, there was
no difference in insulin concentration between the three trials, and
thus the increased FSR during the PRO-CHO trial compared with
the two CHO-only trials would seem to be related more to the
increased availability of amino acids.
With regard to whole body protein balance, several previous
studies utilized leucine tracer methodology (12, 14, 15), and all
showed that net protein balance was positive only when protein
was added to a CHO beverage compared with CHO alone (12,
14, 15). Levenhagen and colleagues (14, 15) attributed the
106 • APRIL 2009 • www.jap.org
1400 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE

Values are means ⫾ SE in ␮mol/l; n ⫽ 6; aP ⬍ 0.05 vs. L-CHO and H-CHO at same time point. bP ⬍ 0.05 vs. L-CHO at same time point. cP ⬍ 0.05 vs. H-CHO at same time point. dPRO-CHO ⬎ L-CHO and
positive net balance to increased whole body synthesis and no

293⫾10a

175⫾13a

208⫾17a

181⫾13a

220⫾14a
Total AAd,f 2,469⫾65 2,539⫾57 2,213⫾55 2,012⫾65 1,958⫾174 2,574⫾85 2,215⫾171 2,426⫾188 1,971⫾78 1,817⫾34 2,184⫾167 2,482⫾103 3,053⫾352b 2,683⫾59a 2,738⫾68a
221⫾31

129⫾10
327⫾44
224⫾17
57⫾4a

106⫾7a

218⫾9a

54⫾3a

58⫾1a
67⫾4
44⫾4

54⫾3

8⫾1

84⫾2
4h change in breakdown, whereas our data are similar to Koop-

H-CHO (main effect treatment; P ⬍ 0.05). ePRO-CHO ⬎ H-CHO (main effect treatment; P ⬍ 0.05). fMain effect for time (P ⬍ 0.05). See Table 1 for definitions of L-CHO, H-CHO, and PRO-CHO trials.
man et al. (12) in that there was no change in synthesis
(represented by NOLD in the present study) and a decrease in
a

190⫾13a

202⫾17a

163⫾10a

208⫾12a
breakdown. The discrepancies may be explained in part by

147⫾19
284⫾13

241⫾34
322⫾48
226⫾15
59⫾3a

98⫾6a
160⫾9a

54⫾2a

61⫾2a
66⫾4
47⫾3

60⫾3

8⫾1

81⫾2
2.5 h

methodological differences. First, Levenhagen et al. (14, 15)

had subjects ingest a single bolus dose and measured changes
after 3 or 6 h of recovery, while in the present study and that
of Koopman et al. (12), subjects received smaller doses at
a

77⫾13a

118⫾13a
197⫾22a
192⫾30a

229⫾28a

162⫾17a

237⫾23a
309⫾29

61⫾20

284⫾69
356⫾31
289⫾64

94⫾10
170⫾19
Pro-CHO

56⫾4a

73⫾8a
69⫾7

63⫾6

8⫾1
frequent intervals over 3– 4 h of recovery. The total amount of
1h

protein ingested also differed between studies; Levenhagen

et al. (14, 15) had subjects ingest 10 g of protein with 8 g CHO
and 2 g fat, whereas Koopman et al. (12) had subjects ingest
312⫾40b

193⫾12c
265⫾12

232⫾42

230⫾27

152⫾28
79⫾4a
62⫾5
41⫾5
66⫾6

56⫾3

139⫾6
133⫾9
8⫾1
50⫾3

78⫾7

131⫾8
61⫾3
179⫾4
beverages at a rate of 0.7 g CHO ⫹ 0.25 g protein䡠kg⫺1 䡠h⫺1,
30 min

or ⬃ 18 g protein/h based on the average mass of their subjects,

for 6 h of exercise and 4 h of recovery. Subjects in the present
study ingested substantially more protein than these other
290⫾16b
256⫾23

228⫾43

224⫾33

100⫾10

174⫾17

140⫾17

141⫾11
54⫾7
39⫾7
55⫾6

50⫾3
47⫾5
90⫾8

8⫾1
43⫾3

70⫾7

112⫾8
55⫾5
studies (12, 14, 15), and further examination of the dose
0h

response of protein ingestion on protein balance after endur-

ance exercise is warranted.
229⫾35
301⫾48

96⫾12

99⫾10

83⫾10

While the changes in whole body protein balance during

200⫾8
52⫾4
42⫾5
36⫾3

220⫾7
47⫾3
21⫾3
51⫾5

9⫾2
34⫾2

62⫾3
121⫾5
76⫾3
30⫾3

recovery in the present study are comparable to data from

4h

Koopman et al. (12), those authors provided nutrition through-

out the exercise session and recovery, whereas subjects in the
212⫾10

245⫾35
295⫾55

101⫾12

137⫾20

100⫾10
57⫾4
45⫾7
43⫾4

230⫾8
48⫾3
28⫾3
58⫾7

9⫾1
37⫾2
119⫾9
67⫾4

86⫾6
39⫾3

present study were only fed during recovery. A novel finding

2.5 h

from the present study was that whole body protein synthesis
decreased from 1 to 4 h of recovery regardless of type of
nutrients ingested. This suggests a possible temporal relation-
251⫾18

71⫾22

287⫾57
329⫾62
244⫾16

119⫾15

170⫾17

186⫾33
115⫾10

144⫾12
61⫾5

56⫾5

51⫾2
47⫾3
97⫾8

9⫾1
44⫾2

81⫾7

54⫾5

ship between the effects of exercise on whole body protein

H-CHO

1h

synthesis during recovery from aerobic exercise. However,

lacking a measurement of resting whole body protein synthe-
sis, we could not resolve whether this represented a return to
306⫾26b
244⫾27

50⫾13

256⫾49

224⫾24

100⫾16

153⫾11

137⫾15

141⫾12
57⫾7

57⫾6

51⫾3
50⫾4
96⫾6

8⫾1
39⫾4

74⫾7

108⫾9
54⫾5
30 min

baseline levels or a potential decrease below resting values.

One might question the apparent disconnect between the whole
body and muscle protein synthetic responses. However, mixed
muscle FSR was calculated based on the postexercise response
295⫾19

267⫾28
361⫾63
253⫾12

135⫾20
137⫾22

185⫾11

169⫾12

159⫾11
64⫾4
46⫾5
61⫾5

51⫾4
55⫾3

8⫾1
42⫾5

85⫾3

126⫾6
65⫾3

over 4 h, whereas the leucine balance data was divided into

0h

“early” and “late” time bins. Thus, while whole body rates
declined so too might have the mixed muscle FSR, but we did
not obtain biopsies throughout the recovery period to evaluate
208⫾20

62⫾23

201⫾26
369⫾81
237⫾21

117⫾13

139⫾14
54⫾3

35⫾2

52⫾5
21⫾2
52⫾5
93⫾9
9⫾1
35⫾2

66⫾6

80⫾4
31⫾1
88⫾8

this directly. It is possible that the data are congruent, but one
4h

measure has the temporal resolution to show an early and a late

response, whereas the other does not. In addition, mixed
218⫾14

192⫾30
376⫾69
220⫾12

muscle FSR accounts for only ⬃25% of whole body protein

57⫾3
53⫾8
41⫾2

50⫾3
28⫾3
60⫾4
95⫾8
8⫾1
39⫾3
126⫾9
66⫾3
140⫾9
89⫾3
39⫾2
105⫾8
2.5 h

synthesis (19), and it could be that the magnitude or direction

of change in other proteins is not consistent with that seen for
skeletal muscle. We also report that the attenuation in breakdown
242⫾18

196⫾39
364⫾61
226⫾11

107⫾11

155⫾14

148⫾11
L-CHO

58⫾5
43⫾6
53⫾4

54⫾5
45⫾3
89⫾4

8⫾1
43⫾2

71⫾2

108⫾3
53⫾2
142⫾7

during PRO-CHO declined during recovery, resulting in lower

1h

positive net balance at 4 h compared with 1 h postexercise. While

the net balance was still positive compared with that to the
284⫾21

218⫾34
428⫾70

121⫾11

180⫾15

181⫾22

CHO-only trials, it occurred while the blood amino acid concen-

Table 3. Blood amino acids

63⫾4
53⫾8
59⫾4

245⫾9
58⫾4
53⫾3
102⫾4

8⫾1
48⫾2

78⫾3

127⫾5
62⫾3
161⫾8
30 min

trations remained high, and thus amino acid availability cannot be

the only factor involved in the attenuation in breakdown.
Whole body leucine oxidation was increased in the PRO-
284⫾21

206⫾32
416⫾63
241⫾14

116⫾12

184⫾13

167⫾12

159⫾11
65⫾5
45⫾5
57⫾4

52⫾4
52⫾4
100⫾6

8⫾1
46⫾2

78⫾2

124⫾4
61⫾3

CHO trial compared with both CHO-alone trials, which is

0h

comparable to the results of both Koopman et al. (12) and

Levenhagen et al. (14). In addition, the present study showed
that the oxidation of leucine was higher at 4 h compared with
1 h of recovery. The increased oxidation at 4 h of recovery
Argd,f

Leud,f
Lysd,f

Phed,f

Thrd,f
Tyrd,f
Vald,f

occurred when the leucine concentration was high in both the

Prod,f
Iled,f
Hise
f

Asn
Asp

Met

Serf
Tau
Glu
Gln
Gly
Ala

blood and muscle. It is plausible that the increase in leucine

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 NUTRITION AND MUSCLE PROTEIN KINETICS AFTER AEROBIC EXERCISE
concentration increased activation of muscle branched-chain
oxoacid dehydrogenase complex (BCOAD), which regulates
BCAA oxidation, and is known to increase in activity with an
increase in leucine (7). Our results are also comparable to
Wilkinson et al. (29), who showed an increase in whole body
leucine oxidation during late recovery (3 h) compared with
early recovery (45 min) with the ingestion of a CHO-EAA
beverage.
With regard to our second hypothesis, we found that adding
protein or additional CHO to a drinking strategy that provided
1.2 g CHO䡠kg⫺1 䡠h⫺1 did not enhance muscle glycogen syn-
thesis during recovery. Several investigators have suggested
this rate of CHO ingestion is the threshold for maximizing
glycogen synthesis during recovery (6, 10) but no previous
study has tested this proposal by directly comparing 1.2 g
CHO䡠kg⫺1 䡠h⫺1 vs. a higher rate of CHO ingestion. The drinks
in the present study were formulated from a 17–22% glucose
polymer solution that subjects ingested at a rate of 750 ml/h,
which is similar to that provided in other studies (11, 27, 28).
One discrepancy from some previous studies is that we did not
detect a significant difference in plasma insulin between trials,
although insulin tended to be higher during PRO-CHO com-
pared with the two CHO-only conditions. However, higher
blood insulin levels do not necessarily translate into a higher
muscle glycogen resynthesis rate, especially when large
amounts of CHO are ingested (10, 11). For example, Jentjens
et al. (11) reported no difference in glycogen resynthesis rate
when they compared ingestion of 1.2 g CHO vs. 1.2 g CHO ⫹
0.4 g protein, despite higher insulin in the latter trial. This
suggests that other factors such as the rate of gastric emptying
and intestinal absorption of ingested glucose, glucose entry
into the muscle, or glycogen synthase activity may have been
limiting (10). Finally, it is worth noting that there were modest
but significant increases in GI distress scores during recovery
in the present study, which were mainly due to a sensation of
severe bloating in half of our subjects after 3– 4 h. This finding
was consistent with the opinion of some authors (6, 10), who
have proposed that very high rates of CHO ingestion may not be
well tolerated by all athletes (10). From a practical standpoint,
consideration should therefore be given to potential GI discomfort
and the potential ramifications in an applied setting (e.g., on
volitional exercise performance after short-term recovery).
In summary, we have shown that ingesting protein with
CHO during recovery from prolonged endurance exercise in-
creased muscle FSR and improved whole body net protein
balance compared with CHO alone. However, adding protein
or additional CHO to a drinking strategy that provided 1.2 g
CHO䡠kg⫺1 䡠h⫺1 did not further augment glycogen synthesis.
The higher FSR during recovery could promote muscle adap-
tation during recovery from acute exercise by stimulating the
synthesis or repair of proteins that facilitate energy provision
and force production. Additional studies are warranted to
resolve the specific population(s) of muscle proteins that are
stimulated by protein and CHO ingestion after aerobic-based
exercise, e.g., myofibrillar vs. mitochondrial.
ACKNOWLEDGMENTS
We thank Todd Prior for assistance with experimental trials and data
analyses, Jason Tang for assistance with the GCMS protocol, and our subjects
for their time and effort.
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Copyright © 2009 American Physiological Society. All rights reserved.
1401
GRANTS
This project was supported by an operating grant from the Natural Sciences
and Engineering Research Council of Canada (NSERC). K. R. Howarth was
supported by a NSERC Graduate Scholarship, and N. A. Moreau held a
Canadian Institutes of Health Research Graduate Scholarship.
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