Protein Ingestion before Sleep Improves
Postexercise Overnight Recovery
PETER T. RES1, BART GROEN1, BART PENNINGS1, MILOU BEELEN1, GARETH A. WALLIS2,
ANNEMIE P. GIJSEN3, JOAN M. G. SENDEN3, and LUC J. C. VAN LOON1,3
1
Department of Human Movement Sciences, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University
Medical Centre+, Maastricht, THE NETHERLANDS; 2GlaxoSmithKline Nutrition, Brentford, UNITED KINGDOM;
and 3Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University
Medical Centre+, Maastricht, THE NETHERLANDSABSTRACTRES, P. T., B. GROEN, B. PENNINGS, M. BEELEN, G. A. WALLIS, A. P. GIJSEN, J. M. G. SENDEN, and L. J. C. VAN LOON.
Protein Ingestion before Sleep Improves Postexercise Overnight Recovery. Med. Sci. Sports Exerc., Vol. 44, No. 8, pp. 1560–1569, 2012.
Introduction: The role of nutrition in modulating postexercise overnight recovery remains to be elucidated. We assessed the effect ofprotein ingestion immediately before sleep on digestion and absorption kinetics and protein metabolism during overnight recovery from asingle bout of resistance-type exercise. Methods: Sixteen healthy young males performed a single bout of resistance-type exercise in theevening (2000 h) after a full day of dietary standardization. All subjects were provided with appropriate recovery nutrition (20 g ofprotein, 60 g of CHO) immediately after exercise (2100 h). Thereafter, 30 min before sleep (2330 h), subjects ingested a beverage with(PRO) or without (PLA) 40 g of specifically produced intrinsically [1-13C]phenylalanine-labeled casein protein. Continuous intravenous
infusions with [ring-2H5]phenylalanine and [ring-2H2]tyrosine were applied with blood and muscle samples collected to assess protein
digestion and absorption kinetics, whole-body protein balance and mixed muscle protein synthesis rates throughout the night (7.5 h).
Results: During sleep, casein protein was effectively digested and absorbed resulting in a rapid rise in circulating amino acid levels,
which were sustained throughout the remainder of the night. Protein ingestion before sleep increased whole-body protein synthesis rates(311 T 8 vs 246 T 9 KmolIkgj1 per 7.5 h) and improved net protein balance (61 T 5 vs j11 T 6 KmolIkgj1 per 7.5 h) in the PRO vs the
PLA experiment (P G 0.01). Mixed muscle protein synthesis rates were È22% higher in the PRO vs the PLA experiment, which reachedborderline significance (0.059%Ihj1 T 0.005%Ihj1 vs 0.048%Ihj1 T 0.004%Ihj1, P = 0.05). Conclusions: This is the first study to showthat protein ingested immediately before sleep is effectively digested and absorbed, thereby stimulating muscle protein synthesis andimproving whole-body protein balance during postexercise overnight recovery. Key Words: SLEEP, RECOVERY, EXERCISE,
MUSCLE, STABLE ISOTOPES, CASEIN

APPLIED SCIENCES

R

increase in protein breakdown (11,28) but does not affectprotein synthesis rates (10,21,28). Intake of protein and/or
amino acids is required to stimulate postexercise protein
synthesis thereby allowing protein balance to become positive (8,12,23,28,32,36). In accordance, dietary protein administration with or without CHO before (35,38), during (3),and/or immediately after exercise (23,28,32,35,38) has beenshown to stimulate net muscle protein accretion during
postexercise recovery.
In general, most studies assess the effect of food intake on
the muscle protein synthetic response to exercise in an
overnight fasted state (10,12,21,23,26,28,32,35,36). Under
these conditions, it seems reasonable to assume that thelimited endogenous availability of amino acids from the gut
and/or the intramuscular free amino acid pool prevents a
substantial rise in postexercise muscle protein synthesis rate.
Such postabsorptive conditions differ from normal everyday
practice in which recreational sports activities are often
performed in postprandial conditions, such as in the evening.
Recently, we evaluated the effect of exercise in the evening
and the efficacy of protein ingestion immediately after exercise on protein synthesis during subsequent overnightesistance-type exercise training represents an effective strategy to augment muscle protein accretion(13,31). A single session of resistance-type exercise
stimulates both protein synthesis and breakdown rates(6,31,37). Although resistance-type exercise stimulates protein synthesis to a greater extent than protein degradation,
protein balance will remain negative in the absence of nutrient intake (6,8,31). Consequently, both exercise and nutrition are required to obtain a positive protein balance,
allowing muscle hypertrophy to occur. CHO ingestion during postexercise recovery attenuates the exercise-induced

Address for correspondence: Luc J.C. van Loon, Ph.D., Department of Human
Movement Sciences, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre+, PO Box 616, 6200 MD
Maastricht, The Netherlands; E-mail: L.vanLoon@maastrichtuniversity.nl.
Submitted for publication September 2011.
Accepted for publication January 2012.
0195-9131/12/4408-1560/0
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DOI: 10.1249/MSS.0b013e31824cc363

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recovery (4). We observed an increase in protein synthesis
during the first few hours of postexercise recovery when
protein was ingested. However, muscle protein synthesis
rates during subsequent overnight sleep were found to be
unexpectedly low with values being even lower than thoseobserved in the normal basal postabsorptive state (4). It was
speculated that the lack of an increase in plasma amino acidand/or insulin concentration prevents muscle protein synthesis rates to be elevated throughout the night.
The present study was undertaken to assess whether provision of dietary protein before sleep leads to adequate dietary
protein digestion and absorption thereby increasing plasmaamino acid availability. In addition, we assessed whether such a
proposed increase in plasma amino acid availability throughout
the night would stimulate protein synthesis and/or attenuate
protein breakdown, thereby improving protein balance during
overnight recovery from exercise. It was hypothesized that theprovision of dietary protein before sleep could be an effective
nutritional intervention to increase plasma amino acid availability, stimulate skeletal muscle protein synthesis, and, as such,
improve whole-body protein balance during overnight sleep.
Therefore, 16 recreationally active young males werestudied during overnight recovery from a single bout of
resistance-type exercise performed in the evening after afull day of dietary standardization. Subjects were provided
with a bolus of protein or placebo (PLA) immediately before sleep after which protein digestion and absorption kinetics and subsequent overnight muscle protein synthesis rates
were assessed during sleep. For this purpose, a specifically
produced intrinsically [1-13C]phenylalanine-labeled protein
was provided. Combined with continuous intravenous infusion of [ring-2H5]phenylalanine and [ring-2H2]tyrosine, thisallows direct assessment of in vivo dietary protein digestion
and absorption kinetics, whole-body protein balance, andmuscle protein synthesis rates during overnight sleep. This is
the first study to assess the effect of protein ingestion immediately before sleep as a dietary strategy to stimulate
muscle protein synthesis and, as such, to improve proteinbalance during postexercise overnight recovery.

METHODS

SPORTS NUTRITION AND OVERNIGHT RECOVERY

Age (yr)
Height (m)Weight (kg)BMI (kgImj2)
Body fat (%)
Trunk fat (%)
Leg fat (%)
Lean mass (kg)Leg volume (L)1RM leg press (kg)
1RM leg extension (kg)Physical activity (hIwkj1)

PRO (n = 8)

PLA (n = 7)

22.9 T 0.7
1.81 T 0.02
74.2 T 2.3
22.7 T 0.6
13.7 T 0.9
11.8 T 0.8
15.2 T 0.9
63.9 T 1.6
9.0 T 0.4
201 T 9133 T 6
6.3 T 1.1

22.4 T 0.8
1.84 T 0.02
76.3 T 2.6
22.5 T 0.8
13.0 T 0.9
11.5 T 0.9
13.8 T 0.9
66.4 T 2.3
9.5 T 0.3
220 T 15132 T 9
5.2 T 1.0

Values are expressed as means T SEM.
PRO, protein experiment; PLA, placebo experiment; BMI, body mass index.

sured with a digital balance with an accuracy of 0.1 kg (SecaGmbH, Hamburg, Germany). Body composition (fat andfat-free mass) was determined by a dual-energy x-ray
absorptiometry scan (Hologic, Inc., Bedford, MA). With thedual-energy x-ray absorptiometry scan, the lean mass andpercent body fat were determined on a whole-body level andfor specific regions (trunk and legs). Leg volume was determined by anthropometry as described by Jones andPearson (20). All subjects participated in a familiarization
trial where a safe lifting technique was demonstrated andpracticed for the two exercises (leg press and leg extension).
Thereafter, maximum strength was estimated using themultiple-repetition testing procedure (27). In another session,at least 1 wk before the first experimental trial, subjects’ one
repetition maximum (1RM) was determined (25). After warming up, the load was set at 97.5% of the estimated 1RM andincreased after each successful lift until failure. Five-minuterest periods were allowed between attempts. A repetition was
regarded valid when the subject used proper form and wasable to complete the entire lift in a controlled manner without assistance.
Diet and physical activity. All subjects received thesame standardized meal the evening before the test and astandardized diet throughout the subsequent experimental
day (0.16 MJIkgj1Idj1, consisting of 57 Energy percentage(En%) CHO, 13 En% protein, and 30 En% fat). The energy
content of the standardized diet was based upon energy
requirements estimated using the Harris–Benedict equation(18) and adjusted using a physical activity factor of 1.6 toensure ample energy intake (29). Breakfast, lunch, and dinner were provided at the laboratory and were eaten under
supervision. During the experimental day, participants
ingested 1.2 gIkgj1 protein via the standardized diet with anadditional 40 g (0.54 T 0.01 gIkgj1) of protein provided in
the PRO experiment. All subjects were instructed to refrainfrom exhaustive physical labor and exercise and to keep
their diet as constant as possible 2 d before the experimental
day. Food intake and physical activity questionnaires werecollected for 2 d before the experiment.
Experimental protocol. An overview of the experimental protocol is given in Figure 1. At 1630 h, subjects
reported to the laboratory where, at 1645 h, a standardized

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Subjects. A total of 16 recreationally active men wereselected to participate in this study. One trial in the PLA
experiment had to be excluded because of a problem with acatheter that obstructed isotope infusion. Subjects were randomly assigned to either the protein (PRO: n = 8) or theplacebo experiment (PLA: n = 7) experiment. Subjects’characteristics are presented in Table 1. All subjects were
fully informed on the nature and possible risks of the experimental procedures before their written informed consent was
obtained. The study was approved by the Medical EthicalCommittee of the Maastricht University Medical Centre, The
Netherlands.
Pretesting. All subjects participated in two screening
sessions, separated by at least 7 d. Body weight was mea-

TABLE 1. Subjects’ characteristics.

APPLIED SCIENCESFIGURE 1—Protocol for the measurement of the effect of protein ingestion before sleep on subsequent overnight recovery. A drink with 60 g of CHO
and 20 g of whey protein was provided immediately after exercise. Subjects were randomly assigned to receive either 40 g of casein protein or placebo(water only) at 2330 h.

meal was provided. At 1830 h, a Teflon catheter was inserted into an antecubital vein for the primed continuous
infusion of [ring-2H5]phenylalanine and [ring-2H2]tyrosine.
A second Teflon catheter was inserted in the contralateral handvein for arterialized blood sampling. Blood was arterializedby placing the hand in a hotbox (1), and a resting/backgroundblood sample was drawn. After a background blood sample
was collected (t = j4.5 h), the primed continuous tracer
infusion was started (1900 h), and subjects rested in a supine
position for 1 h. HR was monitored using a portable HR
monitor (Polar Electro Oy). The exercise protocol was started
at 2000 h and consisted of a bilateral lower limb resistancetype exercise workout with eight sets of eight repetitions on
the horizontal leg press machine (Technogym BV, Rotterdam,
The Netherlands) and eight sets of eight repetitions on the leg
extension machine (Technogym BV). On both machines,
one set was performed at 55% of the individual 1RM, one
was performed at 65%, and six were performed at 75%.
There was a resting period of 2 min between sets and a
5-min rest between exercises. The entire protocol requiredÈ45 min to complete. All subjects were verbally encouraged
during the test to complete the whole protocol. Water wasprovided ad libitum during exercise. After the exercise protocol (2100 h, t = j2.5 h), subjects remained in a supine
position, and a blood sample from the arterialized handvein was obtained. Drinks providing 60 g of CHO and 20 gof whey protein (Lucozade Sport Body Fuel and Lucozade
Sport Recovery; GlaxoSmithKline Nutrition, Brentford,
United Kingdom) were ingested immediately after exercise.
At 2330 h, a muscle biopsy from the vastus lateralis muscle
was taken, after which subjects received the PRO or the PLA
drink. Subsequently, subjects remained in a supine position
until 2400 h. Thereafter, subjects slept for 7 h, after which
participants were awoken the next morning at 0700 h fora second muscle biopsy. Blood samples (8 mL) were takenfrom the arterialized hand vein at t = j4.5, j3.5, j2.5, j2,
j1.5, j1, j0.5, and 0 h; 15, 30, and 60 min; and 1.5, 2.5,
3.5, 4.5, 5.5, 6.5, and 7.5 h after test drink ingestion. Muscle
biopsies were taken at t = 0 and 7.5 h after PRO or PLA
ingestion.

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PRO administration. Subjects ingested 450 mL waterwith (PRO) or without (PLA) 40 g of intact intrinsically[1-13C]phenylalanine-labeled casein protein. Intrinsically[1-13C]phenylalanine-labeled casein protein was obtained byinfusing a Holstein cow with large quantities of [1-13C]phenylalanine, collecting milk, and purifying the casein fraction as
13
described previously (39). The [1- C]phenylalanine enrichment in the casein fraction averaged 6.1 mole percent excess(MPE). The casein protein met all chemical and bacteriological specifications for human consumption. All solutions were flavored by adding strawberry flavor andartificial sweetener (GlaxoSmithKline).
Stable isotope tracers. The stable isotope tracers [ring2
H5]phenylalanine and [ring-2H2]tyrosine were purchased from
Cambridge Isotope Laboratories, Inc. (Andover, MA), anddissolved in 0.9% saline before infusion. Continuous intravenous infusion (during a period of 12 h, 0.06 KmolIkgj1Iminj1of [ring-2H5]phenylalanine, 0.023 KmolIkgj1Iminj1 of [ring2
H2]tyrosine) of the labeled isotopes was performed using
a calibrated IVAC 598 pump (San Diego, CA). Both the phenylalanine and tyrosine pools were primed (2.4 KmolIkgj1of [ring-2H5]phenylalanine, 0.925 KmolIkgj1 of [ring2
H2]tyrosine) such that whole-body phenylalanine kinetics
could be calculated using established tracer models (17,33,34).
Infusates were prepared by the pharmacy of the Maastricht
University Medical Centre.
Muscle biopsies. Muscle biopsies were obtained fromthe middle region of the vastus lateralis muscle (15 cm above
the patella) and approximately 3 cm below the entry throughthe fascia with the percutaneous needle biopsy technique described by Bergstrom (5). The first muscle biopsy was takenfrom the right leg. The biopsy taken 7.5 h after test drink
ingestion was collected from the left leg. All muscle samples
were carefully freed from any visible fat and blood, rapidly
frozen in liquid nitrogen, and stored at j80-C until subsequent analysis.
Plasma analysis. Blood samples (8 mL) were collected
in EDTA-containing tubes and centrifuged at 1000g and
4-C for 10 min. Aliquots of plasma were frozen in liquid nitrogen and stored at j80-C until analysis. Plasma glucose

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SPORTS NUTRITION AND OVERNIGHT RECOVERY

kinetics and the fractional synthetic rate (FSR) of mixed
muscle protein. Total, exogenous, and endogenous phenylalanine rate of appearance (Ra) and plasma availability of
dietary phenylalanine (i.e., the fraction of dietary phenylalanine that appeared in the systemic circulation (Pheplasma))were calculated using modified Steele equations adapted forstable isotope methodology (16,34). These parameters were
calculated as follows:
Ra ¼

Exo Ra ¼

F  pV½ðC2 þ C1 Þ = 2 ½ðEiv2  Eiv1 Þ = ðt2  t1 ÞðEiv2 þ Eiv1 Þ=2

Total Ra ½ðEpo2 þ Epo1 Þ = 2 þ V ½ðEpo2  Epo1 Þ = ðt2  t1 Þ
Eprot
Endo Ra ¼ Total Ra  Exo Ra  FAUCExoPheRa
Pheplasma ¼ 100
Pheprot

where F is the infusion rate (KmolIkgj1 body weight
Iminj1), pV is the distribution volume for phenylalanine(125 mLIkgj1) (17), C1 and C2 are the phenylalanine
concentrations (mmolILj1), and Eiv1 and Eiv2 are the plasma[ring-2H5]phenylalanine enrichments (expressed in MPE) inarterialized plasma at times 1 (t1) and 2 (t2), respectively.
Exo Ra represents the plasma entry rate of dietary phenylalanine, Epo is the plasma phenylalanine enrichment for theoral tracer, and Eprot is the [1-13C]phenylalanine enrichment
in the ingested protein; PheProt is the amount of phenylalanine ingested, and AUCExoPheRa represents the area under the
curve of ExoPheRa, which corresponds to the amount of
dietary phenylalanine that appeared in the blood during a
7.5-h period after protein ingestion.
The total rate of disappearance (Rd) of phenylalanine equals
the rate of phenylalanine hydroxylation and utilization forprotein synthesis. These parameters can be calculated as follows:
Rd ¼ Ra  VC2  C1t2  t1

Because whole-body Rd comprises the rate of phenylalanine
disappearance from the free amino acid pool in the blood
due to protein synthesis and oxidation, whole-body protein
synthesis can be calculated as Rd minus oxidation. Whole-bodyphenylalanine oxidation can be determined from the conversion (hydroxylation) of [ring-2H5]phenylalanine to [ring2
H4]tyrosine. The rate of hydroxylation (Q pt) was calculated
by using the following formula (33):
Qpt ¼ Qt

Et
Ep



Qp
F þ Qp

where Qt and Qp are the flux rates for [ring-2H4]tyrosine and[ring-2H5]phenylalanine, respectively; Et and Ep are the [ring2
H4]tyrosine and [ring-2H5]phenylalanine enrichments in plasma, respectively; and F is the infusion rate of phenylalanine.

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concentrations were analyzed with the Cobas Fara semiautomatic analyzer (Uni Kit III, 07367204; Roche, Basel, Switzerland).
Insulin was analyzed by radioimmunoassay (Linco Human
Insulin RIA kit; Linco Research, Inc., St. Charles, MO).
Plasma (100 KL) for amino acid analyses was deproteinizedon ice with 10 mg of dry 5-sulfosalicylic acid and mixed, andthe clear supernatant was collected after centrifugation. Plasmaamino acid concentrations were determined by High Performance Liquid Chromatography, after precolumn derivatization
with o-phthalaldehyde. For plasma [1-13C]phenylalanine,[1-13C]tyrosine, [ring-2H5]phenylalanine, [ring-2H4]tyrosine,and [ring-2H2]tyrosine enrichment measurements, plasma
phenylalanine and tyrosine were derivatized to theirt-butyldimethylsilyl derivatives, and their 13C and/or 2H
enrichments were determined by electron ionization gas
chromatography–mass spectrometry (Agilent 6890N GC/
5973N MSD; Little Falls, NJ) using selected ion monitoring of masses 336, 337, and 341 for unlabeled and [1-13C]and [ring-2H5]-labeled phenylalanine, respectively. We
applied standard regression curves in all isotopic enrichment analyses to assess linearity of the mass spectrometer andto control for the loss of tracer. Enrichments (MPE) were calculated according to Biolo et al. (7) to correct for the presenceof both the [1-13C] and [ring-2H5]phenylalanine isotopes.
Muscle analyses. For measurement of [ring-2H5]phenylalanine enrichment in mixed muscle protein, 55 mg ofwet muscle was freeze dried. Collagen, blood, and othernon–muscle fiber material were removed from the muscle
fibers under a light microscope. The isolated muscle fiber
mass (2–3 mg) was weighed, and eight volumes (8  dryweight of isolated muscle fibers  wet–dry ratio) of ice-cold
2% perchloric acid were added. The tissue was then homogenized and centrifuged. The PRO pellet was washed withthree additional 1.5-mL washes of 2% perchloric acid, dried,and hydrolyzed in 6-M HCl at 120-C for 15–18 h. The hydrolyzed protein fraction was dried under a nitrogen stream
while it was heated to 120-C; then, a 50% acetic acid solution was added, and the hydrolyzed protein was passed overa Dowex exchange resin (AG 50W-X8, 100-200 mesh hydrogen form; Biorad, Hercules, CA) using 2 M of NH4OH.
The eluate was divided over two vials for the measurement of [ring-2H5]phenylalanine enrichment in mixed
muscle protein. The latter was determined by derivatizing[ring-2H5]phenylalanine to N-(tert-butyldimethylsilyl)-Nmethyltrifluoroacetamide-phenyl-ethylamine. Thereafter,
the ratios of labeled/unlabeled derivatives were determined by gas chromatography–mass spectrometry. Standard regression curves were applied to assess linearity ofthe mass spectrometer and to control for loss of tracer.
The coefficient of variance for the measurement of [ring2
H5]phenylalanine enrichment in mixed muscle protein averaged 1.0% T 0.1%.
Calculations. Ingestion of [1-13C]phenylalanine andintravenous infusion of [ring-2H5]phenylalanine and [ring2
H2]tyrosine with muscle and arterialized blood sampling
were used to simultaneously assess whole-body amino acid

Whole-body protein synthesis was calculated using thefollowing:
PRO synthesis ¼ Total Rd  Phe hydroxylation
Phe net balance ¼ PRO synthesis  Endo Ra

Fractional rate of mixed muscle protein synthesis (FSR)
was calculated by dividing the increment in enrichment in
the product, i.e., protein-bound [ring-2H5]phenylalanine, by
the enrichment of the precursor. Muscle FSR (%Ihj1) was
calculated as follows (23):
FSR ¼

$Ep
 100
Eprecursor t

APPLIED SCIENCES

where $Ep is the delta increment of PRO-bound [ring2
H5]phenylalanine during incorporation periods, Eprecursor
is the average plasma [ring-2H5]phenylalanine enrichment
during the period for determination of amino acid incorporation, and t indicates the time interval (h) between biopsies.
Statistics. All data are expressed as means T SEM.
ANOVA was used to determine the effects of treatment as a
between-subjects factor and time (t = 0–7.5 h) as a withinsubjects factor, as well as any interaction between the two.

In case of a significant interaction, group differences wereanalyzed for each time point separately (i.e., multiple t-tests
using Bonferroni correction). For non–time-dependent variables, an unpaired Student’s t-test was used to compare
differences in between groups. All calculations were performed using SPSS 15.0 (SPSS, Inc., Chicago, IL). Statistical significance was set at P G 0.05.

RESULTS
Participants. Subjects’ characteristics are presented inTable 1. No differences in age, weight, height, body fat, legvolume, 1RM leg press, 1RM leg extension, and habitualphysical activity were observed between groups. All
subjects were able to sleep during their overnight stay andreported no problems sleeping. The use of a catheter extension cable allowed us to draw blood samples throughout thenight without waking the subjects. Average HR throughout
the night did not differ between groups (54 T 3 and 53 T
1 bpm in the PRO and PLA experiments, respectively).
Plasma metabolites. Plasma glucose concentrations
changed over time in both groups (P G 0.01). However, notreatment  time interactions were observed for plasma glucose
concentration. Plasma glucose concentrations throughout the

FIGURE 2—Plasma phenylalanine, tyrosine, leucine, and total essential amino acid concentrations. Values are expressed as means T SEM. Data wereanalyzed with repeated-measures ANOVA (treatment  time). Plasma phenylalanine: treatment effect, P G 0.01; time effect, P G 0.01; interaction oftreatment and time, P G 0.01. Plasma tyrosine: treatment effect, P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. Plasma
leucine: treatment effect, P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. Plasma total essential amino acids: treatment effect,
P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. *Significantly different from PLA (P G 0.05). PRO, protein experiment;
PLA, placebo experiment.

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night averaged 5.1 T 0.1 and 5.2 T 0.1 mmolILj1 in the
PRO and PLA experiments, respectively, with no significant differences between experiments. Plasma insulin levels
showed a significant treatment  time interaction (P G 0.01).
In the PRO experiment, plasma insulin concentrations werehigher at t = 15 and 30 min when compared with PLA(P G 0.01), with values reaching 20.8 T 2.1 mUILj1.
Throughout the night, plasma insulin concentrations did
not differ significantly between experiments (12.1 T 0.9
and 10.8 T 0.8 mUILj1 in the PRO and PLA experiments,
respectively). Plasma essential amino acid concentrations
increased after ingestion of the recovery nutrition that wasprovided immediately after cessation of exercise in bothgroups (P G 0.01) after which they returned to baseline
values within 150 min. After supplement ingestion before
sleep, total plasma essential amino acid concentrations increased rapidly and remained elevated throughout the nightin the PRO experiment when compared with the PLA experiment (P G 0.01). Plasma essential amino acid levels
showed a significant treatment  time interaction. Plasma
phenylalanine, tyrosine, leucine, and total essential aminoacid concentrations over time are presented in Figure 2.

Plasma tracer enrichments. Significant time 
treatment interactions were observed for all plasma tracer
enrichment data. Plasma [1-13C]phenylalanine enrichments
increased after protein ingestion in the PRO group after whichlevels remained elevated in the PRO compared with the PLA
group throughout the night (P G 0.05; Fig. 3A). Plasma[ring-2H5]phenylalanine, [ring-2H4]tyrosine, and [ring-2H2]tyrosine enrichments decreased after protein ingestion in the
PRO group. The enrichments of these three tracers weresignificantly lower in the PRO group compared with the PLA
group for 2.5, 3, and 4 h, respectively, after ingestion of thetest drinks (P G 0.05; Figs. 3B, C, D).
Whole-body PRO metabolism. Whole-body PRO
kinetics were calculated for the overnight recovery period. A
significant treatment  time interaction was observed for
total phenylalanine rate of appearance, exogenous phenylalanine rate of appearance, endogenous phenylalanine rateof appearance, and total phenylalanine Rd. Total phenylalanine rate of appearance (Ra) was higher in the PRO comparedwith the PLA group between t = 0.5 h and t = 3.5 h (P G 0.05;
Fig. 4A). Exogenous phenylalanine Ra was greater in the
PRO compared with the PLA group throughout overnight

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APPLIED SCIENCESFIGURE 3—Plasma [1-13C]phenylalanine (A), [ring-2H5]phenylalanine (B), [ring-2H2]tyrosine (C), and [ring-2H4]tyrosine (D) enrichments. Values are
expressed as means T SEM. Data were analyzed with repeated-measures ANOVA (treatment  time). Plasma [1-13C]phenylalanine enrichment:
treatment effect, P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. Plasma [ring-2H5]phenylalanine enrichment: treatment
effect, P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. Plasma [ring-2H2]tyrosine enrichment: treatment effect, P G 0.01; timeeffect, P G 0.01; interaction of treatment and time, P G 0.01. Plasma [ring-2H4]tyrosine enrichment: treatment effect, P G 0.01; time effect, P G 0.01;
interaction of treatment and time, P G 0.01. *Significantly different from PLA (P G 0.05). PRO, protein experiment; PLA, placebo experiment.

APPLIED SCIENCESFIGURE 4—Total phenylalanine rate of appearance (Ra) (A), exogenous phenylalanine Ra (B), endogenous phenylalanine Ra (C), and total phenylalanine Rd (D). Values represent means T SEM. Data were analyzed with repeated-measures ANOVA (treatment  time). Total plasma phenylalanine
Ra: treatment effect, P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. Exogenous plasma phenylalanine Ra: treatment effect,
P G 0.01; time effect, P G 0.01; interaction of treatment and time, P G 0.01. Endogenous plasma phenylalanine Ra: treatment effect, P G 0.01; time effect,
P G 0.01; interaction of treatment and time, P G 0.01. Plasma phenylalanine Rd: treatment effect, P G 0.01; time effect, P G 0.01; interaction of treatment
and time, P G 0.01. *Significantly different from PLA (P G 0.05). PRO, protein experiment; PLA, placebo experiment.

FIGURE 5—A, Rates of whole-body protein breakdown, synthesis, and oxidation rates and net protein balance (expressed as micromoles of phenylalanine per kilogram per 7.5 h) in the PRO and PLA experiments measured during the 7.5 h of overnight recovery. B, Mixed muscle protein FSR
during overnight recovery (0–7.5 h) in the PRO and PLA experiments using average plasma [ring-2H5]phenylalanine enrichment as a precursor. A
tendency toward higher FSR values in the PRO versus the PLA experiment was observed during overnight recovery. Values represent means T SEM.
Data were analyzed with an unpaired Student’s t-test. *Significantly different from PLA (P G 0.05); †different from PLA (P = 0.05). PRO, protein
experiment; PLA, placebo experiment.

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recovery (P G 0.05; Fig. 4B). In the PRO group, 50% T 2% of
the ingested protein had become available as plasma-derived
amino acids during the 7.5-h postprandial phase. Endogenous
phenylalanine Ra was higher in the PRO compared with the
PLA group at t = 0.5 h (P G 0.05; Fig. 4C). Total phenylalanine Rd was higher in the PRO compared with the PLA
group between t = 0.5 h and t = 3.5 h (P G 0.05; Fig. 4D).
Whole-body phenylalanine hydroxylation rates were similar
between groups. Whole-body protein synthesis rates werehigher in the PRO versus the PLA experiment resulting in a
net PRO balance of +61 T 5 versus j11 T 6 KmolIkgj1 per
7.5 h, respectively (P G 0.01; Fig. 5A).
Muscle analyses. Mixed muscle PRO FSR, with mean
plasma [ring-2H5]phenylalanine enrichment as the precursor,
was calculated for the 7.5 h of overnight sleep (Fig. 5B).
Muscle FSR during overnight recovery showed È22% higher
values in the PRO compared with the PLA experiment, whichreached borderline significance (0.059%Ihj1 T 0.005%Ihj1
vs 0.048%Ihj1 T 0.004%Ihj1, respectively, P = 0.05).

DISCUSSIONSPORTS NUTRITION AND OVERNIGHT RECOVERY

Medicine & Science in Sports & Exercised

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APPLIED SCIENCES

The present study examined the effect of dietary protein
ingestion immediately before sleep on dietary protein digestion and absorption kinetics and subsequent protein metabolism during overnight recovery from a single bout of
resistance-type exercise. The main findings are twofold: dietary protein ingested immediately before sleep 1) is effectively digested and absorbed, thereby increasing overnightplasma amino acid availability, and 2) stimulates muscle protein
synthesis rates, thereby improving overnight protein balance.
We hypothesized that dietary protein ingested immediately before sleep is effectively digested and absorbed,
thereby increasing plasma amino acid availability throughout the night. To assess this, we applied specifically produced intrinsically [1-13C]phenylalanine-labeled casein (39).
This allowed the assessment of dietary protein digestion andabsorption kinetics in vivo in humans (24,39). Consistent
with our hypothesis, we observed a substantial rise in circulating plasma amino acid concentrations after the ingestion of 40 g of intrinsically labeled casein that was sustained
throughout overnight recovery (Fig. 2). These plasma aminoacids were derived from the ingested protein as is evident
from the increase in both plasma phenylalanine concentration (Fig. 2A) and plasma [1-13C]phenylalanine enrichmentlevel (Fig. 3A). In line with previous findings during daytime (30), we show that about approximately half of theingested casein becomes available as plasma amino acids
throughout overnight recovery. The present study is the firstto provide data showing that dietary protein ingested immediately before sleep is digested and absorbed normallythroughout the night and, therefore, represents an effective strategy to increase amino acid availability during
overnight sleep.
An increase in plasma amino acid concentration has previously been shown to stimulate protein synthesis and in-hibit protein breakdown, thereby improving net proteinbalance (12,28,29,36). In line with these observations, weobserved substantially higher whole-body protein synthesis
rates during the night after dietary protein ingestion before
sleep (PRO) when compared with the control experiment(PLA). Because protein ingestion before sleep did not affectwhole-body protein breakdown rates, net protein balance
became positive in the PRO experiment (+61 T 5 vs j11 T
6 KmolIkgj1 per 7.5 h in the PRO vs the PLA experiment,
respectively; Fig. 5A).
Because any change at the level of the skeletal muscle
cannot be accurately determined from whole-body protein
kinetics, we also assessed muscle protein FSR by collecting
muscle biopsies before and after postexercise overnight recovery (Fig. 5B). Overnight FSR values were 22% higher in
the PRO compared with the PLA experiment, reachingborderline statistical significance (0.059 T 0.005 vs 0.048 T
0.004, respectively, P = 0.05). Because muscle protein
synthesis is only stimulated for several hours after an increase in plasma amino acid availability (2,9), it could bespeculated that differences in FSR values between the PRO
and PLA experiments were greatest during the first 4 h of
postprandial sleep. Muscle biopsy sampling between 0300
and 0400 h would have provided us with appropriate muscle
free phenylalanine precursor enrichment data and a likely
higher muscle protein synthesis rate during this specific timeframe. However, muscle biopsy collection during this timeframe would not have been possible without waking the
subjects. Therefore, we assessed protein balance and muscle
protein synthesis rates throughout the overnight period, withplasma phenylalanine enrichments as a reliable precursor
pool. Furthermore, we chose a parallel study design, despite
its obvious limitations, to prevent confounding from theappearance of [1-13C]phenylalanine administered during
the first experiment on the follow-up experiment in the same
individual. Regardless, this study clearly shows that protein
ingested immediately before sleep is normally digested andabsorbed, thereby stimulating overnight muscle protein
synthesis rates. Phillips et al. recently demonstrated that
acute postexercise changes in muscle protein synthesis andbreakdown rates can be predictive of the adaptive response
to more prolonged intervention (19,40). In line with thesefindings, it will be of importance to assess whether the acutemetabolic benefits conferred from the application of dietary
protein supplementation immediately before sleep willtranslate to a greater skeletal muscle adaptive response to
more prolonged resistance-type exercise training.
Besides the proposed relevance to optimize skeletal
muscle reconditioning during prolonged resistance-type exercise training in a sport-specific application, nighttime
protein supplementation might also be of substantial clinicalrelevance. For example, because the muscle protein synthetic response to food intake has been reported to be blunted in the elderly (14,15,22), it is speculated that nighttime
protein provision could prove to be an effective nutritional
strategy to combat age-related muscle loss by providing anadditional anabolic stimulus. In general, such a feeding
strategy could be of important clinical relevance in many
compromised clinical (sub)populations.
In conclusion, this is the first study to show that protein
ingested immediately before sleep is effectively digested andabsorbed, thereby stimulating muscle protein synthesis andimproving whole-body protein balance during postexercise
overnight recovery.

This research was funded by a research grant from GlaxoSmithKline.
The study’s ClinicalTrials.gov ID is NCT00991523.

The authors’ responsibilities were as follows: P.T.R., G.A.W., and
L.J.C.V.L. designed the study; P.T.R. organized and carried out
the clinical experiments with the assistance of B.P., B.G., M.B., andJ.M.S.; A.P.G. and J.M.S. performed the stable isotope measurements. P.T.R. performed the (statistical) analysis of the data andwrote the article together with L.J.C.V.L.; B.G., B.P., M.B., andG.A.W. reviewed the article. B.G. and M.B. provided medical assistance. G.A.W. was a researcher with GlaxoSmithKline Nutrition,
Brentford, United Kingdom, at the time of the experiments.
The authors thank Thijs Käfer, Sam Ballak, Arnoud Aldenkamp,
and Antoine Zorenc for their assistance in supporting the experiments and the enthusiastic subjects who volunteered to participate
in these experiments.
None of the authors had a conflict of interest.
The results of the present study do not constitute endorsementby the American College of Sports Medicine.

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APPLIED SCIENCES

SPORTS NUTRITION AND OVERNIGHT RECOVERY

Medicine & Science in Sports & Exercised

Copyright © 2012 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.

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