Experimental Gerontology 82 (2016) 58–66

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Experimental Gerontology

journal homepage: www.elsevier.com/locate/expgero

Leucine-enriched protein supplementation does not influence

neuromuscular adaptations in response to a 6-month strength training
programme in older adults☆
Séverine Stragier a,b, Stéphane Baudry a, Jacques Poortmans b, Jacques Duchateau a, Alain Carpentier a,b,⁎
a
Laboratory of Applied Biology and Neurophysiology, Université libre de Bruxelles (ULB), 808 Route de Lennik, 1070 Brussels, Belgium
b
Laboratory for Biometry and Exercise Nutrition, Université libre de Bruxelles (ULB), 808 Route de Lennik, 1070 Brussels, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: This study investigated the effect of protein supplementation enriched in leucine on muscle hypertrophy and
Received 6 January 2016 strength gain in response to a 6-month strength training programme of the plantar flexor muscles in older adults
Received in revised form 2 June 2016 (aged over 60 yr). To this end, changes in neuromuscular parameters were compared between two groups
Accepted 6 June 2016
(matched for age, sex and initial maximal strength) that trained twice a week for 24 weeks, with one group re-
Available online 07 June 2016
ceiving a protein supplementation enriched with leucine (supplementation group, n = 12), and another group
Section Editor: Christiaan Leeuwenburgh receiving a placebo (control group, n = 13) that provided subjects with a similar energy intake that the protein
supplementation. Muscle thickness and pennation angle of gastrocnemius medialis were used as indices of mus-
Keywords: cle hypertrophy. Maximal strength and associated electromyographic activity of leg muscles, and voluntary acti-
Electromyography vation (assessed by the interpolated twitch technique) were recorded. Peak torque in response to 3-pulse train
Twitch delivered to the tibial nerve was also recorded. After training, muscle thickness (+8.4%) and pennation angle
Ultrasonography (+13.4%) were increased, regardless of training groups (p b 0.05). Maximal plantar flexion strength (+28.2%)
Voluntary activation and voluntary activation (+29.5%) increased to the same extent for both training groups. Peak torque evoked
by 3-pulse train increased similarly between training groups (+13.4%, p b 0.001). Regardless of protein supple-
mentation, results obtained in twelve subjects indicate that muscle hypertrophy mainly occurred in the last
12 weeks of the programme. Our results suggest that protein supplementation enriched in leucine neither poten-
tiated the neuromuscular adaptations to strength training, nor influenced the time course of these adaptations in
older adults.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
In young adults, the gain in muscle mass in response to strength
training can be potentiated when combined with protein supplementa-
tion (ACSM, 2009; Vieillevoye et al., 2010), likely reflecting the cumula-
tive effect of exercise (Phillips et al., 1999; Tipton et al., 2003) and
protein intake (Miller et al., 2003; Paddon-Jones et al., 2005) on protein
synthesis. Indeed, it is well accepted that muscle hypertrophy is
achieved from successive periods of positive protein balance, which
occurs when protein are ingested after a strength training session
(Campbell and Leidy, 2007). The mTOR pathway is a main contributor
of muscle protein synthesis (Dreyer et al., 2008), and can be activated
☆ The authors declare no competing financial interests.
⁎ Corresponding author at: Laboratory for Biometry and Exercise Nutrition, Faculty for
Motor Sciences, Université libre de Bruxelles, 808, Route de Lennik, CP 640, 1070
Brussels, Belgium.
E-mail address: acarpent@ulb.ac.be (A. Carpentier).
by leucine, an essential branch chain amino acid (Lynch et al., 2003;
Tokunaga et al., 2004).
In contrast to young adults, protein supplementation seems to be
less efficient to increase muscle protein synthesis in older adults when
combined with exercise (Fiatarone et al., 1994; Godard et al., 2002;
Verdijk et al., 2009). This may reflect the age-associated alterations of
muscle protein metabolism in response to exercise (Fry et al., 2011;
Kumar et al., 2009) and protein ingestion (Pannemans et al., 1998;
Welle et al., 1994). It has been shown greater proportion of leucine is re-
quired to optimally stimulate the rate of muscle protein synthesis in
healthy older adults (Katsanos et al., 2006). However, only one study
has investigated the influence of enriched-leucine supplementation
combined with a 12-week strength training programme in older adults
(Trabal et al., 2015). The results indicated a trend for greater strength
gain for the enriched-leucine supplementation group compared with a
control group although no difference was observed for muscle mass,
suggesting the strength gain relied mainly on neural adaptations. How-
ever, the influence of protein supplementation on the respective contri-
bution of muscular and neural adaptations in strength gain has not been

http://dx.doi.org/10.1016/j.exger.2016.06.002
0531-5565/© 2016 Elsevier Inc. All rights reserved.
 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66
investigated so far. Furthermore, even though gains in muscle mass can
be observed after only five weeks of training in young adults (Seynnes
et al., 2007), the lower rate of hypertrophy (Welle et al., 1994) and
the reduced effect of protein supplementation in older adults (Verdijk
et al., 2009) may require longer time to benefit from protein supple-
mentation in healthy older adults.
Therefore, the current study investigated whether protein supple-
mentation enriched in leucine might potentiate muscle hypertrophy
and strength gain in response to a 24-week strength training
programme in older adults. To discard the influence of the additional di-
etary energy induced by protein supplementation, two groups of sub-
jects trained twice a week for 24 weeks, with one group receiving a
protein supplementation enriched with leucine (supplementation
group, n = 12), and another group receiving a placebo (control group,
n = 13) that provided subjects with a similar energy intake that the
protein supplementation. We hypothesized that protein supplementa-
tion should induce greater changes in muscle architecture (used as an
index of muscle hypertrophy; Aagaard et al., 2001; Kawakami et al.,
1995) and a larger increase in muscle strength in response to electrical
stimulation. In a subgroup of subjects, we also investigated the influence
of protein supplementation on the time course of muscle and neural ad-
aptations to know whether leucine-enriched supplementation may
modify the respective contribution of neural and muscular factors in
strength gain.
2. Methods
2.1. Subjects
Thirty five older subjects (21 women and 14 men, see Table 1)
volunteered to participate in this study. All the subjects were communi-
ty-dwelling older adults who are moderately active and who did not re-
port any health conditions. All subjects signed an informed consent and
underwent a medical check-up prior to their participation in the study.
Approval for the project was obtained from the local Ethics Committee,
and all procedures used in this study conformed to the Declaration of
Helsinki.
Subjects were not engaged in any strength training programme for
at least 6 months prior to their participation to the study and were
instructed to maintain their habitual activity outside the prescribed
strength training programme for the entire duration of the study. Indi-
viduals with hepatic or renal function disorders, Parkinson's disease,
multiple sclerosis, diabetes, stroke or cardiac history were excluded
from this study. Volunteers who had orthopaedic problems within
12 months prior to the study were not enrolled. Subjects were assigned
in one of the three following groups: two groups performed a strength
training programme (2 sessions a week) for 24 weeks, with one group
receiving a protein supplementation enriched with leucine (supple-
mentation group, n = 12), and another group receiving a placebo (con-
trol group, n = 13). A third group was composed of 10 subjects who did
not train but received the placebo (no-training group) to show that
even after a long period (6 months) the variables of interest remained
rather constant in our group of older adults. An attempt was made to
match the three groups for sex, age, and initial strength of leg muscles
(Table 1). Each subject participated in at least two experimental ses-
sions lasting 2–3 h (one session before and one session after the 24-
Table 1
Subjects characteristics of the control group and training groups (with supplementation or placebo).
Group Age (years)
Control (n = 10; 7 women) 68.8±4.5
Training + supplementation (n = 12; 7 women) 63.3±3.1
Training + placebo (n = 13; 7 women) 63.2±3.0
Values are means ± SD.
59
week period). The experimental sessions were performed within one
week before and after the training programme. One additional experi-
mental session was performed by 12 subjects randomly selected for
whom all the variables could be recorded, especially the level of volun-
tary activation that was somehow uncomfortable (6 of each training
group) after 12 weeks of training (mid-term) to document the time
course of muscular and neural adaptations. A total of 25 subjects com-
pleted the 6 months training with a compliance to training sessions of
98.6%.
2.2. Dietary intakes
Dietary intake was assessed by a food survey conducted over 7 con-
tinuous days before the training period. The participants were clearly
instructed to maintain their normal eating pattern and report detailed
information about each food item as accurately as possible including
composition, preparation and portion size. For the latter, they were
asked to weigh the items before cooking using their personal weighting
scales. When this was not possible, food quantities were estimated
using food models. Diets were analysed by Nutrilog Software (Nutrilog
version 2.6, France). Nutrilog software was implemented for specific
local food based on a Belgian nutritional database (Nubel, 4th edition).
Biochemical assessment of protein nutritional status was approached
by nitrogen balance before training. Total urinary nitrogen was used to-
gether with total protein intake (g protein/6.25) to estimate nitrogen
balance (NBAL = Nin − Nout). Subjects were asked to complete two
24-h urine collections (encompassing 1 week day and 1 weekend
day). During each 24-h epoch, dietary intake was analysed to calculate
the balance between nitrogen intake and excretion. Urine volume was
measured to the nearest millilitre, and aliquots were stored at −20 °C,
as well. Total nitrogen analysis was then performed on each urine sam-
ple with the Kjeldahl method (Lynch and Barbano, 1999) (Table 1). Par-
ticipants were asked to maintain the same diet and physical activity
throughout the study period.
2.3. Nutritional supplementation
Both placebo and protein groups received two dietary supplementa-
tions per day (108 kcal each). Protein supplementation was a flavoured
powder composed of 20 g of milei hydrolysat (with 1.9 g of leucine),
enriched with 0.6 g of leucine and 7 g of maltosweet. Placebo supple-
mentation contained 27 g of maltosweet and the same artificial sweet-
ener as for the protein supplement. Both supplements were dissolved in
200 ml water and consumed with breakfast and dinner on the non-ex-
ercise days, and with breakfast or dinner and immediately after com-
pleting the training session on the exercise days.
2.4. Surface electromyogramme
The surface electromyogramme (EMG) signals were recorded from
soleus, gastrocnemius medialis, gastrocnemius lateralis and tibialis an-
terior of the right leg with surface electrodes (silver-silver chloride elec-
trodes of 8-mm diameter) placed in a bipolar configuration with an
inter-electrode distance (centre to centre) of 2 cm. Before attaching
the electrodes, the skin was shaved when necessary and cleaned with
a solution of alcohol, ether, and acetone to reduce the impedance at
Height (cm) Weight (kg) Protein intake Nitrogen
g kg−1 balance gN
167.2±6.3 66.9±8.9 1.29 ± 0.17 4.34 ± 3.07
168.9±6.1 68.4±13.3 1.23 ± 0.34 1.05 ± 2.60
168.5±17.1 71.3±16.0 1.08 ± 0.30 2.87 ± 5.56
60 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66
the skin-electrode interface. The electrodes were filled with gel and at-
tached longitudinally over each muscle belly with adhesive tape. The
electrodes for the soleus were placed 3 cm below the muscle-tendon
junction of the gastrocnemius medialis in line with the Achilles tendon.
The electrodes for gastrocnemii were placed midway between the fem-
oral condyle and the respective muscle-tendon junction. The electrodes
for tibialis anterior were placed at one third of the distance between the
fibular head and the lateral malleolus, and 1 cm lateral to the tibial crest.
The reference electrodes were placed over the tibia. The EMG signals
were amplified (1000×) and band-pass filtered (10–1000 Hz) prior to
be A/D sampled at 2 kHz (Power 1401, 16-bit resolution, Cambridge
Electronic Design, UK) and stored on a computer.
2.5. Electrical nerve stimulation
Electrical stimuli (1-ms duration) applied to the tibial nerve were
delivered via a constant current stimulator (DS7A, Digitimer,
Hertfordshare, UK) that was connected to two surface electrodes (sil-
ver-silver chloride electrodes of 8-mm diameter) attached to the skin
with adhesive tape at the knee level of the right leg. The cathode was
placed in the popliteal fossa and the anode located just above the patel-
la. The optimal site of stimulation was determined by moving a pen
electrode (cathode) until the site to elicit an M wave in gastrocnemius
medialis with the largest amplitude at a given intensity was identified.
For all stimulation conditions, the current intensity was set at 150% of
the smallest intensity eliciting a maximal M wave (Mmax) amplitude
in the gastrocnemius medialis to ensure maximal stimulation through-
out the experimental session (Gandevia, 2001).
2.6. Maximal voluntary contraction
The torque developed during a maximal voluntary contraction
(MVC) performed with the ankle plantar flexor muscles of the right
leg was recorded with the subjects seated in a custom-made ergometric
device. The ankle and knee angles were positioned at 90° and 0° (full
knee extension), respectively, and the foot tightly attached to a
footplate that was connected to a force transducer (model 4576A2N,
Kistler, Winterthur, Switzerland). The force transducer signal was am-
plified and low-pass filtered at 200 Hz. Subjects performed at least
three MVCs with the plantar flexor muscles. When the difference in
peak torque of the two greatest MVCs was within 5% of each other,
the greatest value was taken as the maximum. Otherwise, additional tri-
als were performed until the 5% criterion was achieved (all subjects
reached the 5% criterion within 5 MVCs). Moreover, the interpolated
twitch technique was used to assess the level of voluntary activation
of the plantar flexor muscles. Trains of three electrical stimuli (Shield
and Zhou, 2004) triggered 10 ms apart (3-pulse train) were delivered
to the tibial nerve during and immediately after at least two of the plan-
tar flexor MVCs (second and third MVCs), and to the remaining MVCs
when the 5% criterion was not reached (Fig. 1). Three to five MVCs
were also performed with the ankle dorsal flexor muscles in the same
experimental conditions, except that the interpolated twitch technique
was not used.
2.7. Ultrasonography
Muscle architecture of gastrocnemius medialis was assessed from
images obtained with a realtime B-mode echograph (DP-6900Vet,
Shenzhen Mindray Bio-Medical Electronics CO, Ltd., China) with a 6-
cm width linear-array probe (7.5 MHz; 75L60EA) coated with a
water-soluble transmission gel to provide acoustic contact. These
changes were used as indexes of muscle hypertrophy for the plantar
flexor muscles (Aagaard et al., 2001; Kawakami et al., 1995). Measure-
ments were only made on the gastrocnemius medialis, as muscle thick-
ness assessment with ultrasonography for the soleus is not possible due
to its deeper location compared with gastrocnemius medialis, and that
Fig. 1. Torque signal during maximal voluntary contraction (MVC) with superimposed
(ST) and potentiated (PT; triggered at rest after MVC) 3-pulse train (top traces), and
corresponding EMG for the soleus (SOL, mid traces) and gastrocnemius medialis (GM,
bottom traces) muscles before and after the training period for one subject of the
supplementation group.
differences in hypertrophy were not expected between the two gastroc-
nemii. The subjects were seated in the ergometric device with the probe
positioned on the right leg at 30% of the distance between the medial
condyle of the tibia and the centre of medial malleolus, over the mid-
belly of the muscle and oriented along the longitudinal axis of the mus-
cle (Narici et al., 1996). Particular care was taken to reduce pressure on
the skin during image acquisition. Anatomical landmarks were used to
position the probe at the same location before and after the training pe-
riod. Two parameters were measured from each ultrasound scan: mus-
cle thickness and pennation angle of muscle fascicle (Maganaris et al.,
1998; Narici et al., 1996). Images were analysed offline with Image J
software (National Institute of Health, USA).
2.8. Experimental protocol
All subjects participated before and after the training period to one
experimental session consisting of recording muscle architecture, MVC
force and associated EMG, with and without the interpolated twitch
technique. The session began with the ultrasonography recordings
after 10 min of rest in seated posture. Thereafter, the recording and
stimulation electrodes were placed and the stimulus intensity associat-
ed with the Mmax was determined. Then, subjects performed MVCs with
the ankle plantar flexor and dorsal flexor muscles. During and after
MVCs with ankle plantar flexor, electrical stimulus was delivered to
the tibial nerve to assess subject's voluntary activation. The time course
of change in neural and muscular variables was analysed in 12 subjects
(6 per training group) by performing an additional experimental ses-
sion after the 12th week of training (mid-term) during which muscle
architecture, MVC force, and associated EMG and voluntary activation,
and 3-pulse train response were assessed.
2.9. Training programme
The training programmes consisted of two 1-h sessions per week (at
least 48 h apart) over a period of 24 weeks. Each training session began
by a warm up (~10 min) consisting of cycling exercise and ended with
stretching exercises (5 min). The main part of a training session was
composed of two exercises involving the lower leg muscles (leg press
and calf raises with extended knee) performed on a specific machine
(Technogym®). The velocity of movement was not directly controlled
but subjects were asked to perform the exercises at a moderate velocity
 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66
to ensure a correct execution of the movement. The training started
with a total workload of 30 repetitions per exercise (3 sets of 10 repeti-
tions). The initial load corresponded to the maximal load with which a
subject was able to complete 10 repetitions. This load was determined
during the second session (the first session being used to familiarize
the subjects with the exercises) by increasing gradually the load until
subjects could not perform more than 10 repetitions. Four to six sets
were required to determine this load. During the first 24 sessions, the
number of repetitions was progressively increased to reach 50 repeti-
tions per exercise (5 sets of 10 repetitions). In the second part of the
training programme, the total number of repetitions was kept constant
but the number of repetitions within sets was decreased and the num-
ber of sets increased. This was accompanied by an increase in the rela-
tive load lifted (% 10 RM). The rest period between sets was 90–120 s.
Trunk and upper limbs exercises were also performed with machines,
free load, Swiss ball or body weight to increase subject's compliance to
the training programme. These exercises were varied every 5 sessions.
Throughout the training programme, the progressive overload was
achieved by increasing the number of repetition or the load when sub-
jects were able to perform the prescribed training volume with the pre-
scribed load during two consecutive training sessions. Some of the
authors supervised all training sessions.
2.10. Data reduction
Muscle thickness of gastrocnemius medialis was defined as the dis-
tance from superficial aponeurosis to the deep aponeurosis
(Maganaris et al., 1998; Narici et al., 1996), and was measured at two lo-
cations, the left and the right edge of each image. The average of 3 mea-
surements performed on each side was made, and the average of both
sides was used to calculate muscle thickness. Muscle fascicle was de-
fined as a clearly visible fibre bundle lying between the superficial and
deep aponeuroses, and pennation angle was determined as the angle
between the fascicle and its insertion on the deep aponeurosis
(Maganaris et al., 1998; Narici et al., 1996). Two pennation angles
were analysed for each subject, before and after training. Data from 2
subjects (1 subject in each training group) were not used in the analysis
due to technical issues.
Peak torque during each MVC was measured, and the greatest value
was taken as the maximum strength capacity of the subject. The average
value of the rectified EMG (aEMG) was calculated for each muscle dur-
ing a 1-s epoch around peak torque during plantar flexion and dorsal
flexion MVCs. The raw EMG signal from gastrocnemii medialis (n =
25) and lateralis (n = 20), and soleus (n = 15) was normalized to
Mmax peak to peak amplitude to take into account differences in record-
ing conditions between sessions (including the thickness of the volume
conductor between source and detection points; Merletti et al., 2009).
The degree of voluntary activation was calculated as follows: level of
voluntary activation = (1 − ST/PT) ∗ 100, where ST is the torque pro-
duced in response to 3-pulse train triggered during the MVC
(superimposed train), and PT the peak torque developed in response
to the 3-pulse train triggered at rest immediately after the MVC (poten-
tiated train) (Shield and Zhou, 2004). Due to pain sensation induced by
the stimulation, it was not possible to determine voluntary activation in
5 subjects (3 in supplementation training group and 2 in control group).
Therefore, for this variable, data from only 20 out of the 25 subjects en-
rolled in the training groups were used for statistical analysis. In addi-
tion, the peak torque, and maximal rates of torque development and
relaxation were measured from the mechanical response of the 3-
pulse train triggered after the MVC. The level of antagonist coactivation
during plantar flexion MVC was assessed by calculating the coactivation
ratio as follow: coactivation (%) = tibialis anterior EMG during plantar
flexion MVC/tibialis anterior EMG during dorsal flexion MVC ∗ 100. A
similar formula was used to calculated the coactivation ratio during dor-
sal flexion MVC: coactivation (%) = plantar flexors EMG during dorsal
61
flexion MVC/plantar flexors EMG during plantar flexion MVC ∗ 100
(Kellis, 1998).
To detail the mechanisms responsible for changes in maximal
strength in neural and muscular factors, we distinguished neural factors
that correspond to the EMG and voluntary activation, from muscle fac-
tors that correspond to muscle thickness and pennation angle, and the
mechanical response to the 3-pulse train.
2.11. Statistics
The main objective of the study was to determine whether or not
leucine-enriched supplementation potentiated the effect of strength
training. As a consequence, the a priori experimental design consisted
of comparing two groups of individuals performing the same training
but receiving for one group the leucine-enriched supplementation
(supplementation group), and for the other group a supplementation
that brought similar additional dietary energy but did not contain
amino-acids (control group). The effects of the supplementation in
combination with strength training were analysed by 2-way ANOVAs
(supplementation × training). Tukey post-hoc test was used when sig-
nificant interactions were found to determine specific differences be-
tween means. Coefficient of determinations extracted from Pearson
product-moment correlations were calculated for the association be-
tween changes in maximal torque, voluntary activation, muscle thick-
ness and pennation angle. Stepwise, multiple-regression analysis
examined the parameters (voluntary activation, muscle thickness or
pennation angle) that predicted the gain in plantar flexion MVC torque.
In addition, we wanted to control that no change occur when no train-
ing was performed during a similar period (6 months). Comparing this
group (no training) with the supplementation and control groups was
not appropriated as it does not provide relevant information relative
to the main objective of the study (effects of leucine-enriched supple-
mentation combined with strength training). Paired Student t-tests
were therefore used to compare the dependent variables of the un-
trained group. Prior to the comparison of each dependent variable, the
Gaussian distribution of the data was verified by the Kolmogorov-
Smirnov test. The level of statistical significance was set at p ≤ 0.05 for
all comparisons. Values are expressed as mean ± SD in the text and ta-
bles and mean ± SE in the figures.
3. Results
3.1. Control group
No statistical difference was found in the untrained group for the
various studied parameters at 6-month interval (Table 2).
3.2. Training groups
As no statistical difference was observed between the two training
groups after training (p values N 0.05 for supplementation main effect
and supplementation × training interaction), statistical significance
and values represent the training main effect determined by ANOVAs.
3.3. Muscle architecture
Thickness of the gastrocnemius medialis (+8.7 ± 6.7%; p b 0.001)
and pennation angle (+14.3 ± 9.6%; p b 0.001) increased after training
(Fig. 2).
3.4. MVC torque and electrically-induced contractions
Before training, the MVC torque developed by the plantar flexor
muscles was 89.8 ± 28.1 N·m and 93.1 ± 29.3 N·m for the supplemen-
tation and control group, respectively. The MVC torque increased by
similarly in both groups (p b 0.001) after training (supplementation
62 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66

Table 2
Strength, neural and muscular characteristics for the control group (no training, with
placebo).

Before After

Plantar flexor MVC torque (N·m) 101.1±29.3 105.8±28.1

Dorsiflexor MVC torque (N·m) 30.4±7.9 32.7±9.2
Voluntary activation (%) 82.9±21.0 83.3±14.2
aEMG-GM (mV) 0.118±0.080 0.130±0.072
aEMG-GL (mV) 0.115±0.082 0.104±0.061
aEMG-Sol (mV) 0.075±0.045 0.097±0.055
aEMG-TA (mV) 0.103±0.052 0.111±0.057
aEMG-GM/Mmax (%) 2.7±2.2 3.4±2.2
aEMG-GL/Mmax (%) 2.6±1.7 3.4±1.8
aEMG-Sol/Mmax (%) 1.9±1.2 2.5±1.6

3-Pulse train
Peak torque (N·m) 44.9±16.0 47.7±15.1
Rate of torque development (N·m·s−1) 642.3±264.3 664.2±279.2
Rate of torque relaxation (N·m·s−1) 389.5±166.4 388.4±133.9
Mmax (mV) 3.7±2.2 4.5±1.9
Muscle thickness (mm) 17.7±3.5 17.3±3.7
Pennation angle (°) 19.1±4.0 18.8±3.7

Values are means ± SD. MVC: maximal voluntary contraction; aEMG: average value of the
rectified EMG; GM: gastrocnemius medialis; GL: gastrocnemius lateralis; Sol: soleus; TA:
tibialis anterior; EMG-GM/Mmax: aEMG of the gastrocnemius medialis normalized with
the peak to peak amplitude of the maximal muscle compound action potential (Mmax).

group: +33.2 ± 26.4%; control group: 23.7 ± 21.7%) (Fig. 3, top panels).
The level of voluntary activation increased from 76.5 ± 19.4% before
training to 93.2 ± 9.5% after training (p b 0.001) (Fig. 3, bottom panels).

Fig. 3. MVC torque (top panels) and voluntary activation (bottom panels) of the plantar
flexor muscles before (open bars) and after (filled bars) training for the supplementation
group (left panels) and the control group (right panels). Values after training differed
significantly from those recorded before training without differences between groups
(training main effect, p b 0.001), as denoted by *. Data are mean ± SE.

Fig. 2. Muscle thickness (top panels) and pennation angle (bottom panels) of the
gastrocnemius medialis before (open bars) and after (filled bars) training for the
supplementation group (left panels) and the control group (right panels). Values after
training differed significantly from those recorded before training without differences
between groups (training main effect, p b 0.001), as denoted by *. Data are mean ± SE.
The gain in MVC torque was positively associated with the increase in
voluntary activation (r2 = 0.54, p b 0.001; Fig. 4). The aEMG of the gas-
trocnemius lateralis (before: 0.082 ± 0.060 mV; after: 0.120 ±
0.070 mV), the gastrocnemius medialis (before: 0.109 ± 0.077 mV;
after: 0.130 ± 0.061 mV) and the soleus (before: 0.069 ± 0.062 mV,
after: 0.081 ± 0.043 mV) did not change significantly after 6 months
of training (p N 0.05, Table 3). However, the average value of aEMG of
the plantar flexor muscles increased significantly during the MVC after
training regardless of the training group (before: 0.086 ± 0.050 mV;
after: 0.110 ± 0.046 mV; p = 0.02). Furthermore, the aEMG of gastroc-
nemius medialis, normalized to the corresponding Mmax, was increased
after training (before: 2.7 ± 2.2%; after: 3.4 ± 2.2%; p = 0.05). Similar
results were obtained for the gastrocnemius lateralis (before: 2.6 ±
1.7%; after: 3.4 ± 1.8%; p = 0.024) while the aEMG of soleus, normalized
to the corresponding Mmax tended to increase (before: 1.9 ± 1.2%; after:
2.5 ± 1.6%; p = 0.07). For both training groups, the coactivation ratio
during plantar flexion MVC decreased after training (before: 24.5 ±
25.1%; after: 14.0 ± 12.4%; p = 0.023).
Before training, the MVC torque developed by the dorsal flexor
muscles was 28.3 ± 5.1 N·m and 31.0 ± 11.3 N·m for the supplemen-
tation and control group, respectively. The MVC torque increased simi-
larly in both groups (p b 0.001) to reach 34.1 ± 7.0 N·m and 35.6 ±
14.5 N·m after training for the supplementation and the control
group, respectively. The aEMG of the tibialis anterior did not change
 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66 63

3.5. Time course of neural and muscular adaptations

The time course of change in neural and muscular variables was

analysed in 12 subjects (6 per training group). No difference was ob-
served between the two groups and thereby statistical significance
and values represent the training main effect from ANOVAs. MVC torque
increased significantly during the first half (+22.7 ± 21.8% initial value;
p = 0.005) but not during the second half of the training programme
(+6.4 ± 17.3% mid-term value; p = 0.69). Similar results were obtain-
ed for voluntary activation that increased during the first (+ 27.0 ±
42.3% initial value, p = 0.03) but not during the second half of the train-
ing (+3.9 ± 16.0% mid-term value; p = 0.95) (Fig. 5). The mechanical
response to the 3-pulse train did not change statistically in the first half
of the training (+4.1 ± 10.1%; p = 0.19) but increased in the second
half of the training (+ 11.6 ± 9.5; p = 0.005). In contrast, the rate of
torque development tended to increase (p = 0.07) but the rate of
torque relaxation did not change (p = 0.46). Muscle thickness and
pennation angle increased significantly only during the last 3 months
(Fig. 5).

4. Discussion

Fig. 4. Relation between changes in MVC torque and voluntary activation after 24 weeks of
training. The linear regression between these parameters is: y = 1.162x + 1.419 (r2 =
0.54; p b 0.001).
significantly after 6 months of training (before: 0.214 ± 0.129 mV;
after: 0.240 ± 0.110 mV; p = 0.31). The coactivation ratio during dorsal
flexion MVC decreased significantly after training (before: 21.2 ±
13.4%; after: 14.2 ± 7.5%; p b 0.001).
Before training, the peak torque developed in response 3-pulse train
was 50.5 ± 10.0 N·m and 41.3 ± 10.6 N·m for the supplementation and
control group, respectively, and increased similarly (p b 0.001) in the
supplementation (+ 9.0% ± 12.4%) and the control group (+ 14.0 ±
12.1%). The maximal rate of torque development also increased from
570.8 ± 177.8 N·m·s−1 to 654.0 ± 210.6 N·m·s−1 (p = 0.012). In con-
trast, the rate of torque relaxation did not change significantly (before:
374.6 ± 151.3 N·m·s−1; after: 389.1 ± 159.6 N·m·s−1; p = 0.55).
Multiple regression analysis indicates that both voluntary activation
and muscle thickness contribute to the increase in MVC torque of plan-
tar flexor muscles after 24 weeks of training (Strength gains =
19.05 + 0.65 ∗ voluntary activation − 0.25 ∗ muscle thickness; R2 ad-
justed = 0.51 p b 0.05).
The main finding of this study suggests that leucine-enriched protein
supplementation did not potentiated muscle hypertrophy and strength
gain in healthy older adults in response to a 24-week strength training
programme.
4.1. Maximal strength
The training programme led to an increase in MVC strength of the
ankle plantar (+28%) and dorsal flexor muscles (+17%) with no addi-
tional effect of the supplementation. In addition, the torque developed
in response to the brief train of electrical stimuli (3-pulse train delivered
at 100 Hz) increased after training indicating that changes beyond the
neuromuscular junction took place in response to the strength training
programme (Scaglioni et al., 2002). These results are in agreement with
previous work reporting no additional positive effect of protein supple-
mentation on strength gain after lower limb muscles training in older
adults (Leenders et al., 2013; Verdijk et al., 2009; Trabal et al., 2015).
However, compared with the aforementioned studies which investigat-
ed either 12-week strength training with leucine-enriched supplemen-
tation (Trabal et al., 2015), or 12- to 24-week training period with no
enriched leucine-supplementation (Leenders et al., 2013; Verdijk et

Table 3
ANOVA results for all variables in supplementation and control group.

Supplementation effect Training effect Supplementation × training

F-test p value F-test p value F-test p value

Muscle thickness b0.1 (1,20) 0.90 33.5 (1,20) b0.001 b0.1 (1,20) 0.95
Pennation angle 0.7 (1,20) 0.41 53.5 (1,20) b0.001 b0.1 (1,20) 0.86
MVC-PF b0.1 (1.23) 0.96 36.2 (1,23) b0.001 0.5 (1,23) 0.50
MVC-DF 0.1 (1,23) 0.48 22.5 (1,23) b0.001 1.2 (1,23) 0.28
aEMG-GM 0.1 (1,23) 0.73 2.4 (1,23) 0.14 1.4 (1,23) 0.26
aEMG-GL 1.6 (1,23) 0.23 3.1 (1,23) 0.08 0.7 (1,23) 0.41
aEMG-Sol 3.5 (1,23) 0.10 0.52 (1,23) 0.48 b0.1 (1,23) 0.90
aEMG-TA 0.7 (1,23) 0.51 1.5 (1,23) 0.31 0.6 (1,23) 0.55
aEMG-GM/Mmax 0.9 (1,23) 0.35 4.4 (1,18) 0.049 0.2 (1,18) 0.64
aEMG-GL/Mmax b0.01 (1,18) 0.97 4.1 (1,18) 0.024 1.0 (1,18) 0.24
aEMG-Sol/Mmax 2.5 (1,13) 0.13 3.5 (1,13) 0.07 0.3 (1,13) 0.60
Coactivation 0.3 (1,23) 0.58 8.1 (1,23) b0.001 0.6 (1,23) 0.44
PT 3.2 (1,18) 0.09 23.7 (1,18) b0.001 2.6 (1,18) 0.13
PT/dt 1.0 (1,18) 0.34 7.7 (1,18) 0.012 1.7 (1,18) 0.22

MVC: maximal voluntary contraction; aEMG: average value of the rectified EMG; GM: gastrocnemius medialis; GL: gastrocnemius lateralis; Sol: soleus; TA: tibialis anterior; EMG-GM/
Mmax: aEMG of the gastrocnemius medialis normalized with the peak to peak amplitude of the maximal muscle compound action potential (Mmax); EMG-GL/Mmax: aEMG of the gastroc-
nemius lateralis normalized with the peak to peak amplitude of the maximal muscle compound action potential (Mmax). EMG-Sol/Mmax: aEMG of the soleus normalized with the peak to
peak amplitude of the maximal muscle compound action potential (Mmax). PT: peak torque of the three-pulse train; Pt/dt: maximal rate of torque development the three-pulse train.
64 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66
Fig. 5. MVC torque (top left panel) and voluntary activation (bottom left panel) of the plantar flexor muscles, and thickness (top right panel) and pennation angle (bottom left panel) of the
gastrocnemius medialis (bottom panel), before (white bars), after 12 weeks (mid; grey bars) and 24 weeks (black bars) of strength training. Data from the supplementation and control
groups were merged. * and ** denote significant difference between before and mid training values, and between mid and after training values (p b 0.05 and p b 0.01, respectively). Data are
mean ± SE.
al., 2009; Villanueva et al., 2014), we expected that a longer training
duration (6 months) associated with leucine-enriched protein supple-
mentation would have produced greater effects.
4.2. Muscle adaptations
Both pennation angle and muscle thickness of the medial gastrocne-
mius increased after training. These results likely reflect muscle hyper-
trophy in response to training (Aagaard et al., 2001; Kawakami et al.,
1995), but again with no beneficial effect of protein supplementation.
Previous work indicated that compared with young adults, greater pro-
portion of leucine in the protein supplementation is required to opti-
mally stimulate the rate of muscle protein synthesis in older adults
(Katsanos et al., 2006; Rieu et al., 2006). Subjects of the supplemented
group were therefore provided with a 2.5 g leucine associated with
9.6 g of other essential amino-acids that have been shown to stimulate
muscle protein anabolism (Børsheim et al., 2002; Volpi et al., 2003).
However, the ingestion of amino-glucose mixture has been shown to
blunt muscle protein anabolism in older adults, likely due to alterations
in the response of muscle protein synthesis to the glucose-induced en-
dogenous hyperinsulinemia (Volpi et al., 2000). The dietary intake,
composed of protein and carbohydrate used in the present study, may
have reduced the efficacy of the supplementation to increase muscle
mass. Another important factor for the efficacy of protein supplementa-
tion is related to the timing of the intake. Indeed, protein intake imme-
diately after exercise does further improve post-exercise net muscle
protein balance (Esmarck et al., 2001; Tipton et al., 2001). To take this
aspect into account, our subjects had to ingest their supplementation
immediately after the end of each training session. Finally, the lack of
difference in muscle adaptations between the two groups could have
been related to the positive nitrogen balance of our subjects. Previous
work reported that protein supplementation does not potentiate mus-
cle mass induced by strength training when older adults already
ingested adequate amount of protein prior to the strength training pro-
gramme (Campbell and Leidy, 2007). In contrast, Tieland et al. (2012)
observed a gain in lean mass of 1.2 kg after 3 months of training and
1.3 kg after 6 months of training in frail older persons when protein
supplementation was provided. Overall, our results support previous
observation of an age-related decrease in the efficacy of protein supple-
mentation to increase muscle mass combined with strength training
(Leenders et al., 2013), and further suggest that the lack of specific effect
for the combination of strength training and protein supplementation in
older adults does not rely on leucine content. Nonetheless, it is worth
noting that the rather small number of subjects in the supplementation
and control groups may have masked subtle differences in neuromuscu-
lar adaptations.
4.3. Neural adaptations
The only study investigating the influence of enriched-leucine sup-
plementation combined with strength training in older adults observed
a trend for a greater increase in strength in the supplementation group
than in the control group whereas no group difference was observed in
muscle volume (Trabal et al., 2015). This suggests different contribution
of neural and muscular adaptations in strength gain after enriched-leu-
cine supplementation. It is well known that strength gain following re-
sistance training does not parallel muscle hypertrophy, especially in the
early phase of training (Sale, 1988), and neural adaptations seem to play
a greater role in strength gain with advancing age (Häkkinen et al.,
1998; Penzer et al., 2015). In agreement, we observed an increase in
voluntary activation in our two training groups as well as an increase
in aEMG of the gastrocnemius medialis and lateralis (normalized to
Mmax), with a similar trend for the soleus muscle. These results likely
reflect an increased descending drive sent to the motor neurone pools
of the plantar flexor muscles, as supported by the relation between
the gains in voluntary activation and maximal strength. Furthermore,
the decrease in coactivation observed during both plantar flexion and
dorsal flexion MVCs likely reflects neural adaptations contributing to in-
crease the net torque produced at the joint level after training (Carolan
and Cafarelli, 1992). However, leucine-enriched supplementation did
not influence the extent of neural adaptations.
 S. Stragier et al. / Experimental Gerontology 82 (2016) 58–66
4.4. Time course of neural and muscle adaptations
When expressed per session, the gain in MVC torque is ~0.6% which
is similar to the gain reported by Scaglioni et al. (2002) (0.4% per session)
and Häkkinen et al. (2001) (0.6% per session). However, the gain per ses-
sion is 0.9% for the first 12 weeks of training whereas it is only of 0.25%
for the last 12 weeks, indicating that strength enhancement in response
to training mainly occurred at the beginning of the programme. Similar
time course was observed for voluntary activation that increased during
the first part (+27%) but not the last part (+3.9%) of the training pro-
gramme. This is in agreement with an association between the gain in
muscle strength and the increase in voluntary activation in short-dura-
tion (6 weeks) training programme (Penzer et al., 2015). In contrast,
muscle thickness and pennation angle increased only significantly dur-
ing the last 3 months of training (Fig. 2). Accordingly, the peak torque
in response to 3-pulse train did not change during the first half of the
training programme but increased thereafter, suggesting that changes
at the muscle level (hypertrophy) contributed significantly to increase
the maximal force capacity of the muscle in the second part of the train-
ing period. These different time courses in neural and muscular adapta-
tions in response to strength training have been proposed for young
adults (Sale, 1988). The present study indicates that such time courses
occur also in older adults and was not influenced by protein intake. It
is worth noting that due to technical limitations (see Section 2), muscle
mass assessment by ultrasonography was only performed from the gas-
trocnemius medialis. Therefore, it is not possible to rule out the fact that
assessing muscle hypertrophy from one muscle may have hidden earlier
or larger muscle hypertrophy in other leg muscles (McCall et al., 1996).
However, even though muscle thickness and pennation angle may not
be the most sensitive variables to assess muscle hypertrophy, the fact
that changes in these variables, and in the torque evoked by the 3-
pulse train, were only observed in the second half of the training pro-
gramme supports the assumption that muscle adaptations mainly
takes place at a slower rate than neural adaptation. Overall, these results
indicate that despite the well-known reorganization of the neuromuscu-
lar system and the reduced protein synthesis in ageing, underlying
mechanisms of strength improvement are preserved in healthy older
adults, and are not influenced by protein supplementation.
In conclusion, the present results suggest that specific leucine-
enriched protein supplementation does not potentiate neural and
muscular adaptations in response to a 24-week strength training in
older adults.
Acknowledgments
This study was supported by the Walloon Region of Belgium
(Geramino project C6077).
We are indebted to Dr. Simaga Bamodi for performing the medical
examination of the subjects involved in this study.
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