(15-02-14) Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

B. Esmarck, J. L. Andersen*, S. Olsen, E. A. Richter†, M. Mizuno‡ and M. Kjær
Sports Medicine Research Unit, Bispebjerg Hospital, *Copenhagen Muscle
Research Centre, CMRC, Rigshospitalet, †Department of Human Physiology,
University of Copenhagen and ‡Department of Anaesthesia, Righospitalet,
Denmark
(Received 19 December 2000; accepted after revision 17 April 2001)
1. Age-associated loss of skeletal muscle mass and strength can partly be
counteracted by resistance training, causing a net synthesis of muscular
proteins. Protein synthesis is influenced synergistically by postexercise amino
acid supplementation, but the importance of the timing of protein intake
remains unresolved.
2. The study investigated the importance of immediate (P0) or delayed (P2)
intake of an oral protein supplement upon muscle hypertrophy and strength over
a period of resistance training in elderly males. 3. Thirteen men (age, 74 ± 1
years; body mass index (BMI), 25 ± 1 kg m_2 (means ± S.E.M.)) completed a 12
week resistance training programme (3 times per week) receiving oral protein in
liquid form (10 g protein, 7 g carbohydrate, 3 g fat) immediately after (P0) or
2 h after (P2) each training session. Muscle hypertrophy was evaluated by
magnetic resonance imaging (MRI) and from muscle biopsies and muscle strength
was determined using dynamic and isokinetic strength measurements. Body
composition was determined from dual-energy X-ray absorptiometry (DEXA) and
food records were obtained over 4 days. The plasma insulin response to protein
supplementation was also determined.
4. In response to training, the cross-sectional area of m. quadriceps femoris
(54.6 ± 0.5 to 58.3 ± 0.5 cm2) and mean fibre area (4047 ± 320 to 5019 ± 615
µm2) increased in the P0 group, whereas no significant increase was observed in
P2. For P0 both dynamic and isokinetic strength increased, by 46 and 15%,
respectively (P<0.05), whereas P2 only improved in dynamic strength, by 36%
(P<0.05). No differences in glucose or insulin response were observed between
protein intake at 0 and 2 h postexercise.
5. We conclude that early intake of an oral protein supplement after
resistance training is important for the development of hypertrophy in skeletal
muscle of elderly men in response to resistance training.

Ageing is associated with a progressive reduction of skeletal muscle volume
(Lexell et al. 1988) and a concomitant reduction in strength (Grimby & Saltin,
1983; Young et al. 1985; Vandervoort & McComas, 1986; Frontera et al. 1991).
This influences the physical performance and thereby the daily function of the
elderly. However, resistance training has been shown to counteract the atrophy
and loss of strength in this age group (Frontera et al. 1988; Fiatarone et al.
1990; Charette et al. 1991; Welle et al. 1996).
Hypertrophy following resistance training requires net protein synthesis of
the myofibrillar proteins, and hence, a maximal stimulation of protein
synthesis is favourable for the development of muscle hypertrophy in the
elderly. Mixed muscle protein synthesis rate is increased in humans after bouts
of resistance training provided the stimulus is of a sufficient magnitude
(Chesley et al. 1992). This transient increase in protein synthesis is marked
and surpasses the increase in degradation rate, and persists for up to 48 h
following an acute exercise bout (Phillips et al. 1997). Evidence exists in
favour of the balance between protein synthesis and degradation in skeletal
muscle being tipped in favour of protein synthesis by protein intake and
hyperaminoacidaemia during rest (Bennet et al. 1989; Biolo et al. 1997; Smith
et al. 1998), and net protein balance remains negative after training if
individuals remain postabsorptive (Biolo et al. 1995; Phillips et al. 1997).
Yet, amino acid supplementation postexercise has been shown to have a
synergistic effect upon the muscle contraction-induced augmentation of muscle
protein synthesis, when provided both intravenously (Biolo et al. 1997) and
orally (Tipton et al. 1999). The stimulation of protein synthesis after bouts
of resistance exercise probably follows a specific time course. Thus, it has
been observed that protein synthesis is greater 3 h compared to 24 and 48 h
postexercise (Phillips et al. 1997). As protein administration is crucial for
an optimal effect on net protein synthesis, an early intake of protein after
exercise is likely to be important. Recently, it was observed that young
individuals had identical acute protein synthesis responses to an amino acid–
carbohydrate intake during the first hour following ingestion, irrespective of
the supplement being administered 1 or 3 h after resistance exercise (Rasmussen
et al. 2000). However, in a resistance training study on rats the timing of a
mixed meal ingestion after each training session influenced net protein
synthesis over a 10 week training period, as the group that was fed immediately
postexercise increased hindlimb muscle mass more than the group fed 4 h later
(Suzuki et al. 1999). Yet, it is not known whether ingestion of amino acids
immediately postexercise will have a greater effect on the net protein
synthesis compared to a later ingestion in elderly males during a period of
resistance training.
Hence, the purpose of this study was to investigate the importance of the
timing of protein intake after exercise upon the development of muscle
hypertrophy and strength during a period of resistance training in elderly
individuals. Muscle hypertrophy was evaluated by MRI and from muscle biopsy
samples, and muscle strength was determined using both dynamic and isokinetic
strength measurements. The acute glucose, insulin and catecholamine responses
to exercise and supplementation were also determined for 4 h after training.
DISCUSSION The major finding of the present study is that the timing of
protein intake after resistance training bouts in elderly males is of
importance for the development of hyper- trophy in skeletal muscle. Thus, over
a 12 week resistance training period, the CSA of m. quadriceps femoris and MFA-
tot increased by 7 and 22%, respectively, when protein was ingested immediately
after exercise (P0), whereas no significant changes were observed when protein
was supplemented 2 h postexercise (P2) (Figs 2 and 3). The degree of
hypertrophy found in P0 was similar to the findings of other studies
investigating the effects of resistance training in the elderly where no
specific dietary restrictions were reported (Frontera et al. 1988; Brown et al.
1990; Fiatarone et al. 1990; Welle et al. 1996). In addition to the difference
in hypertrophy development between the two groups, it is interesting to note
the absence of any detectable hypertrophy in P2 despite 12 weeks of resistance
exercise identical to the P0 group. This points to the importance of the early
timing of protein intake in recovery from resistance exercise in terms of the
amount of net protein synthesis in skeletal muscle. Such a hypothesis somewhat
resembles the findings of the importance of early carbohydrate intake for the
magnitude of glycogen resynthesis after exercise- induced depletion of muscle
glycogen (Ivy et al. 1988).
The increase in MFA was more pronounced (22%) than that in CSA of the whole
muscle (7%). This is in accordance with the findings of other groups (Frontera
et al. 1988) and indicates that a concomitant reduction in the relative amount
of non-muscular tissue (fat and connective tissue) to CSA takes place in
response to training. This is supported by findings in resistance- trained
fragile elderly women, where a 10% increase in CSA of quadriceps was measured
when corrected for fat and connective tissue versus only a 5% increase when not
corrected for this (Harridge et al. 1999). Furthermore, since the angle of
pennation has been shown to increase with resistance training in young
individuals (Aagaard et al. 2001) it is likely that this contributes to the
discrepancy between the two measurements of the relative increase of the muscle
mass, and thus to the fact that the anatomical CSA of the muscle (determined by
MRI) underestimated the true increase in physiological CSA (determined by
muscle biopsy).
The MFA of fibre-type 1 (MFA-1) was found to be larger than MFA-2 in P-tot (as
well as in P0) in agreement with previous observations in the elderly (Lexell
et al. 1983; Aniansson et al. 1992). However, with training MFA-2 increased
significantly more than MFA-1 in P0, and concomitantly the distribution of MHC-
II increased in P0 (Fig. 4). These changes with resistance exercise are similar
to findings in both young (Andersen & Aagaard, 2000) and elderly individuals
(Charette et al. 1991) and could indicate that training induces a larger
increment in stress of the type 2 fibres than type 1 fibres compared to normal
daily living (Henneman et al. 1965). Further, it was only the area of fibre-
type 2a that increased, as MFA-2b determined histochemically was unaffected by
training. In addition, lean body mass increased more in P0 than in P2 with
training, in agreement with a larger net muscle protein synthesis in P0 over
the 12 week period of resistance training compared to P2.
In line with the observed disparity in the degree of hypertrophy between the
two groups, the muscle strength was also affected differently by the 12 weeks
of training in P0 compared to P2. Thus, isokinetic strength increased at both
measured velocities (60 and 180 deg s_1) in P0, whereas no significant increase
was observed in P2 at any velocity (see Fig. 1B). However, both groups showed
an increase in dynamic training strength assessed as 5 RM (see Fig. 1A), but
this is more likely to reflect the neural factors of learning and coordination
resulting from training (Rutherford & Jones, 1986). Thus, differences between
P0 and P2 were observed only in the isolated non-trained isokinetic knee
extensions, where the increase in strength in P0 was in agreement with other
studies investigating the effect of 12 weeks of resistance training in the
elderly using knee extension as the exercise mode (Frontera et al. 1988; Lexell
et al. 1995).
No significant differences were observed between P0 and P2 for any
anthropometrical, dietary, muscle or strength
Timing of protein intake after resistance exerciseJ. Physiol. 535.1 307
parameter before training (see Table 1). Hence, both P0 and P2 fulfilled the
recommended daily allowance for energy and protein intake (WHO/FAO/UNU, 1985)
and also met the more extensive protein recommendations for the elderly (0.9 g
kg_1 day_1; Campbell et al. 1994). Moreover, in the elderly it has been found
that the protein requirements probably are not increased above normal dietary
intake on non-training days as the myofibrillar protein synthesis was found to
be similar in an exercised leg 23 h postexercise whether high-protein (28 E%)
or isocaloric low-protein meals (7 E%) were ingested (Welle & Thornton, 1998).
Therefore, when subjects are fulfilling the dietary recommendations, it appears
only to be necessary to provide protein supplementation on training days in
order to maximise the net protein synthesis stimulated by the bout of
resistance exercise. However, some controversy exists as to whether or not a
daily nutritional supplement combined with resistance training has an additive
effect on hypertrophy in the elderly. Whereas one group has observed an
additive effect (Meredith et al. 1992), another group did not (Fiatarone et al.
1994). In both studies supplementation was given on top of normal adequate
nutrition, however, and furthermore food recordings in both studies were not
optimal. In the present study subjects in the two groups received the same
controlled protein supplementation per body weight (0.13 g kg_1) and per lean
body mass (0.19 g kg_1) after every training bout. Hence, the findings do seem
to indicate that the timing of the protein intake is of utmost importance for
protein synthesis and muscle hypertrophy.
In the present study muscle protein synthesis was not determined, but it is
evident that the resulting hypertrophy after training is a product of an
accumulation of net synthesis of structural muscle proteins after each
resistance exercise bout. As the synthesis of mixed muscle, myofibrillar and
MHC proteins has been shown to increase in response to resistance training in
the elderly (Yarasheski et al. 1993; Welle et al. 1999; Hasten et al. 2000),
resulting in net protein synthesis over a period of resistance training
(Frontera et al. 1988; Brown et al. 1990; Fiatarone et al. 1990; Welle et al.
1996), it might be surprising that in the present study one of the groups, P2,
did not show any significant increase in muscle CSA. However, two studies have
failed to show an acute response in muscle protein synthesis to resistance
exercise (Tipton et al. 1996; Roy et al. 1997), although in these studies,
trained subjects were used suggesting that the training stimulus could have
been insufficient (Rennie & Tipton, 2000). In the present study all individuals
were untrained prior to the programme, and furthermore relative loads at 75% of
RM were used during the last 6 weeks, making it unlikely that loading was
insufficient to stimulate muscle protein synthesis. Nevertheless, no
hypertrophy was observed in P2 despite the fact that comparable studies with
very
similar (Hakkinen & Hakkinen, 1995) or slightly heavier training protocols
(Frontera et al. 1988; Fiatarone et al. 1990; Welle et al. 1996) have found an
increase in muscle mass. However, we have no well-founded reason for believing
that these studies were carried out in circumstances in which the subjects ate
‘early’ though the changes in muscle mass resemble our findings in P0. Yet,
none of these studies report the dietary habits in association with the
training. Alternatively, it could be speculated that the time of day when the
training was carried out was important. The diurnal hormonal profile could
influence the anabolic response to resistance exercise. Thus, in the present
study the subjects always trained in the morning between 08.00 and 10.00 h with
no differences in the specific time points between the groups P0 and P2. This
was done in order to standardise the conditions, which were best controlled at
this time of the day. Hence, the subjects in the present study may have had a
disadvantage in training compared with other studies, and this could explain
the lack of hypertrophy in P2. Unfortunately, no previous studies report when
the training was carried out; further investigations are required to elucidate
this point.
In spite of this, the difference in hypertrophy between the two groups
suggests that the isolated act of contraction is counteracted by other factors,
e.g. delayed food intake. In line with this, Tipton et al. (1999) have shown
that postexercise net muscle protein balance is negative when individuals are
maintained in the post- absorptive state during recovery, whereas if they
ingest protein and achieve hyperaminoacidaemia the protein balance becomes
positive. Finally, we are confident that both groups trained properly as the
training sessions were always supervised and loads adjusted at every third
training session. This is supported by the increased training strength (5 RM),
which was observed for both groups.
Although not directly determined in this study we do believe that the protein
intake highly stimulated muscle protein synthesis, since in a recent study by
Rasmussen et al. (2000) protein synthesis was elevated 3.5 times above pre-
intake values when a supplement of only 6 g amino acid with 35 g carbohydrate
was ingested, whereas we gave 10 g protein together with 7 g carbohydrate.
Furthermore, with ageing the stimulation of protein synthesis by resistance
exercise has been shown to be preserved (Welle et al. 1994). However, as the
dietary restrictions ended 2 h postexercise the possibility cannot be ruled out
that if P2 regularly ingested a meal shortly after the supplement then the
maximal effective dose of protein was exceeded, hence the stimulatory effect of
the protein supplementation was blunted compared to P0. Yet, the dose–response
relationship of ingested protein and protein synthesis remains to be
elucidated. Consequently, we suggest that the contraction-induced stimulation
of protein synthesis was used to a lesser
B. Esmarck and others308 J. Physiol. 535.1
extent in the formation of muscle protein in P2 compared to P0, provided that
the stimulation of the protein synthesis follows a time course with a rapid
increase within the first few hours following exercise (Phillips et al. 1997).
Moreover, since the amino acid delivery is dependent on blood flow (Biolo et
al. 1995), the intracellular amino acid availability in the exercised muscle
may have been larger in P0 than in P2, hence favouring an increased anabolic
response in P0 as it correlates with intracellular amino acid concentration
(Biolo et al. 1995). Interestingly, Rasmussen and co-workers have found that
protein synthesis and breakdown are stimulated similarly by protein intake in
recovery from resistance exercise whether the protein is ingested 1 or 3 h
after the termination of exercise, at least in young individuals when protein
synthesis in the hour following intake is compared (Rasmussen et al. 2000).
However, a 1 h measurement period may be too short to determine differences
that affect muscle protein synthesis for many hours.
Altogether, our findings suggest that the first 2 h of recovery after
resistance exercise are important for the net protein synthesis during a
strength-training programme evaluated over a period of time, and to optimise
the protein synthesis the intramuscular concentration of free amino acids is
critical if it is not to be a limiting factor.
In the present study muscle fibre hypertrophy was more pronounced in P0 than
in P2 despite identical rises in plasma insulin after the intake of a
supplement containing protein and carbohydrate (Fig. 5). However, it may be
speculated whether the insulin sensitivity of protein turnover is markedly
higher immediately after exercise than 2 h later. This has, however, to our
knowledge not been studied in humans. Yet, the importance of hyperinsulinaemia
with respect to the present study can be questioned. First of all, the effect
of postexercise hyperinsulinaemia has been shown to decrease mixed muscle
protein degradation whereas synthesis is unaffected (Biolo et al. 1999) and,
presumably, the effect is primarily on lysosomal degradation and not
myofibrillar breakdown, which follows on the ubiquitin–proteasome pathway.
Second, a recent study on postabsorptive exercise in diabetic/non- diabetic
rats observed that insulin only played a permissive role at low concentrations
in stimulating protein synthesis (Fedele et al. 2000). Thus, it was concluded
that the effect of insulin on protein synthesis was only apparent in the low
range of plasma insulin, whereas a further increase in insulin did not enhance
net protein synthesis additionally (Fedele et al. 2000).
Finally, we cannot exclude the possibility that our finding of a difference in
hypertrophy between the groups is a result of the relatively low number of
subjects. However, no subjects were systematic outliers and,
furthermore, all subjects in P0 had a larger increase in the relative change
of the CSA of the quadriceps than any subject in P2. Additionally,
theoretically it cannot be ruled out that exercise-induced changes in tissue
and serum levels of anabolic hormones such as growth hormone, cortisol or
insulin-like growth factor 1, which were not determined in the present study,
could contribute to the difference between P0 and P2.
In conclusion, this study investigated the importance of the timing of protein
intake after each exercise bout over 12 weeks of resistance training on
morphological and strength characteristics of skeletal muscle in elderly
individuals. Based on the findings in the present study it appears that the
timing of protein intake after strength training bouts can be important for
protein synthesis and hypertrophy of skeletal muscle in elderly individuals,
and that this appears not to be related to the hyperinsulinaemia in response to
the intake of a protein–carbohydrate supplement. The present findings support
the hypothesis that early intake of protein after resistance exercise enhances
total muscle mass as well as hypertrophy of single muscle fibres in elderly
humans.

Source: Journal of Physiology (2001), 535.1, pp.301–311

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