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Cite journal as: J Gerontol A Biol Sci Med Sci
Sci. 2012 November;67(11):11611169
doi:10.1093/gerona/gls080
The Author 2012. Published by Oxford University Press on behalf of The Gerontological Society of America.
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Advance Access published on March 28, 2012
of Health and Exercise Science and 2Department of Food Science and Human Nutrition, Colorado State University,
Fort Collins.
3Division of Endocrinology, Metabolism, and Diabetes, University of Colorado, Aurora.
We previously showed that consumption of protein immediately after exercise in older adults enhances nitrogen balance
when energy balance (EB) is maintained. Because daily EB routinely varies, it is important to know whether benefits
of postexercise protein consumption also occur with changing EB. Within an experiment, participants consumed an
isonitrogenousisocaloric diet with the timing of a protein (PRO + CHO) or carbohydrate (CHO) beverage immediately
after exercise versus earlier in the day. Within hypocaloric and hypercaloric cohorts, 3-day mean nitrogen balance was not
different when protein was consumed immediately after exercise, although there was a trend (p = .09) for higher nitrogen
balance in the positive EB. However, when data from our three studies were combined, the anabolic effect of postexercise
feeding was evident during positive EB but not negative EB. EB is therefore an important consideration in the postexercise
anabolic effect of protein feeding.
Key Words: NBALExerciseWasting.
Received November 1, 2011; Accepted February 13, 2012
Decision Editor: Rafael de Cabo, PhD
Address correspondence to Benjamin F. Miller, PhD, Department of Health and Exercise Science, Colorado State University,
200 Moby B Complex, Fort Collins, CO 80523-1582. Email: benjamin.f.miller@colostate.edu
21162
MINOR ET AL.
(17,18). Ingestion of amino acids with or without carbohydrate caused a shift toward increased protein balance
postexercise (17,19,20). However, by consuming amino acids with carbohydrates postexercise, net protein balance
was increased to levels greater than if the amino acids or
carbohydrate were ingested alone (19). In the elderly, protein combined with carbohydrate (40-g carbohydrate, 20-g
whey) led to increases in whole-body protein turnover
above those observed with an isoenergetic amount of carbohydrate (60-g carbohydrate; 21).
There is conflicting evidence as to whether timing of protein intake after exercise enhances protein synthesis and
thus LBM (22,23). We have addressed this question by
using NBAL and have shown that in older individuals in EB,
consumption of protein and carbohydrates (chocolate milk)
immediately following moderate aerobic exercise at 55%
VO2max led to greater NBAL than if consumed earlier in the
day (24). Findings from our previous study were interesting
given that differences in NBAL were noted with only the
timing of protein intake differing between trials. Because
many older individuals are habitually in negative EB (25),
and increasing energy intake leads to increases in NBAL
(9,10), we investigated the impact of the timing of protein
intake on NBAL in exercising older individuals in states of
hypocaloric and hypercaloric EB. We hypothesized that
timing protein intake after exercise in states of hypocaloric
and hypercaloric EB would enhance NBAL over values
observed when protein is consumed earlier in the day.
Methods
Study Overview
The study included two separate cohorts, hypocaloric
(n = 10) and hypercaloric (n = 6). In the hypocaloric cohort,
participants were in 15% negative EB for the duration of the
experimental period and participants in the hypercaloric
cohort were in 15% positive EB. Each experiment consisted
of four periods: preexperimental testing, a 7-day lead-in
diet, a 6-day inpatient/experimental period, and post-testing
(Figure 1A). Metabolic information collected during pretesting was used for planning the lead-in and experimental
diets and to determine the relative intensity of the daily
exercise during the experimental period. The 7-day lead-in
diet allowed participants to adapt to the level of dietary
protein that was provided during the experimental period.
All aspects of the study were completed in the same laboratory setting and repeated the design of our previously
published study (24). The study protocol was approved by
the Colorado State University Institutional Review Board
and the Colorado Multiple Institutional Review Board
for human research and adhered to the Declaration of
Helsinki.
Participants
Ten healthy sedentary men (n = 2) and women (n = 8)
participated in the hypocaloric cohort, and six healthy sedentary men (n = 2) and women (n = 4) participated in the
Figure 1. Study timeline (A) and daily timing of meals, exercise, and consumption of beverages during PRO + CHO and CHO trials (B). The timeline in panel B
occurs within the period labeled Experimental in panel A. The order of the PRO and CHO trials was randomized.
11633
EB
EB CHANGES
CHANGES ANABOLIC
ANABOLIC EFFECT
EFFECT
Age (years)
Height (cm)
Weight (kg)
Body mass index (kg/m2)
Body fat (%)
VO2max (mL/kg/min)
Hypercaloric Cohort
Men (n = 2)
Women (n = 8)
Men (n = 2)
Women (n = 4)
All Participants (n = 6)
67 1
183 1
93 1
27.4 0.3
27.9 1.0
32.2 0.5
63 2
165 3*
61 2*
22.3 0.6*
34.5 1.7
28.5 1.7
64 2
169 3
67 4
23.3 0.8
33.2 1.7
29.2 1.4
63 6
178 5
85 3
26.9 2.5
24.7 1.3
29.4 2.1
66 2
160 2*
60 3*
23.5 1.8
37.9 4.2
21.1 1.2*
65 2
166 4
68 6
24.6 1.5
33.5 3.9
23.9 2.0*
Pretesting
Over four separate days, participants completed a series
of pretesting at the Human Performance Clinical/Research
Laboratory at Colorado State University. During the initial
visit, participants completed a Balke protocol graded exercise test on a treadmill under the supervision of a cardiologist. Participants were excluded if the graded exercise test
indicated a cardiac ischemic or hypertensive response to the
exercise. Participants returned to the Human Performance
Clinical/Research Laboratory after an overnight fast, and
resting metabolic rate (RMR; Parvomedics TrueOne 2400,
Sandy, UT) was measured to determine 24-hour resting
caloric expenditure. During the test, participants rested in
the supine position for 45 min and were instructed to remain
still and to refrain from sleeping. The first 15 min of the test
was used to achieve the appropriate flow rate and to allow
participants to become familiarized with the experimental
conditions. The data from the final 30 min of the test were
used to predict RMR. Body composition was then determined using dual-energy X-ray absorptiometry (QDR
4500W, Hologic, Inc., Bedford, MA). On the third day of
testing, participants completed an incremental exercise test
on a cycle ergometer (Monark Excalibur, Groningen, The
Netherlands) with indirect calorimetry (Parvomedics TrueOne 2400) to determine maximal oxygen consumption
(VO2max). Participants pedaled at 50 watts for the first minute,
and watts increased by 20 for women and 30 for men every
2 min thereafter (24). The test was stopped when participants reached volitional exhaustion. After VO2max was
determined, 55% of each participants VO2max was calculated using the American College of Sports Medicine leg
cycle ergometry equation (26).
hypercaloric balance cohort as depicted in Table 1. All participants were nonsmokers between the ages of 55 and
75 years and did not take any medications. Participants
were required to be lactose tolerant because milk was used
as an experimental beverage. Additional exclusion criteria
included obesity (body mass index > 30), recent orthopedic
injury that would impede the ability to exercise, any condition that affected food digestion or absorption, a thyroid
condition (thyroid stimulating hormone <0.05 uU/mL or
thyroid stimulating hormone > 5.0 uU/mL), or any current
illness or infection.
41164
MINOR ET AL.
Experimental/Inpatient Period
The experimental period involved a 6-day inpatient stay
at the University of Colorado Denver CTRC (Figure 1B).
Each participant completed 2 consecutive, 3-day trials in a
randomized crossover design. The diets for each 3-day trial
were reproduced and identical in calorie intake, macronutrients, and foods consumed. The diet plans for Day 1, 2, and
3 were repeated on Day 6, 4, and 5, respectively. The only
difference between trials was the timing of intake of the
protein beverage. For the negative EB cohort, participants
were in a 15% caloric deficit, and similar to the lead-in diet,
the percentage of total kilocalories for each macronutrient
in the inpatient diet was 55% carbohydrate, 30% fat, and
15% protein. Participants in the positive EB cohort were in
positive 15% EB and, similar to the lead-in diet, the inpatient
diet consisted of 1.2-gm protein/kg bw, 30% of calories
from fat, and the balance as carbohydrate.
Participants were admitted to the CTRC the evening
before the 6-day inpatient period and provided dinner as the
final meal of their lead-in diet. The study began the following morning with the start of the study diet and 24-hour
urine collection. Daily weight measurements were recorded
each morning on the same scale after voiding. Day 1 and
Day 6 were spent in a whole-room calorimeter, whereas
days 25 were spent in a patient room on the CTRC. During
days 25, participants were permitted to leave the CTRC
unit twice a day for 30 min. Participants were allowed to eat
only the food that was provided to them by study staff and
to consume water ad libitum. Every day at 4:30 pm, participants completed 1 hour of cycling exercise (Lode, Groningen,
The Netherlands) at 55% of their VO2max. The exercise was
intended to simulate a brisk walk, which was well tolerated
by all participants. Participants were not permitted to engage
in any additional volitional exercise during the day.
Immediately following the daily exercise bout, a postexercise beverage was consumed. The only difference between
the two trials was the timing of intake of the two experimental beverages. In one trial (PRO + CHO), a 248-kcal chocolate milk drink that contained 15.3-gm protein, 43.6-gm
carbohydrate, and 1.3-gm fat (330-gm skim milk, 4-gm
whey protein, and 42-gm chocolate syrup) was consumed
immediately postexercise. In the second trial (CHO), a 247kcal carbohydrate beverage that contained 0.0-gm protein,
63.51-gm carbohydrate, and 0.06-gm fat was consumed
immediately postexercise. During the PRO + CHO trial,
the CHO beverage was consumed as a snack at 10 am and
during the CHO trial, the PRO + CHO beverage was consumed
as a snack at 10 am.
In order to plan the diets for the inpatient period, total
daily EE was estimated. Each participants RMR was multiplied by a separate activity factor for calorimeter and noncalorimeter days. The activity factor was lower for calorimeter
days because ambulatory activity levels are lower when confined to the calorimeter room (E. L. Melanson, PhD, unpublished data, 2010). Exercise EE was estimated from the
steady-state VO2 data (measured during pretesting), and an
additional 20% of exercise calories were added in order to
account for excessive postexercise oxygen consumption.
Participants entered the calorimeter room at 7:45 am on
Day 1 and Day 6 and exited at 7:15 am on the following
morning. The calorimeter room was 12 12 and contained
a bed, sink, toilet, bicycle ergometer, computer, and television.
To prevent air from escaping, all meals were passed through
an air lock that could not be simultaneously opened from the
inside and outside. Daily EE and substrate oxidation were
calculated by the difference in gas content that was entering
and exiting the room. EE and respiratory quotient were
assessed in 1-minute intervals from oxygen consumption
and carbon dioxide production. Gas concentrations were
determined from the flow rate and the differences in CO2
and O2 concentrations between entering and exiting air by
using a fuel-cellbased dual channel O2 analyzer (FC-2
Oxzilla, Sable Systems, International, Las Vegas, NV) and
two infrared CO2 analyzers (CA-10 CO2 analyzers, Sable
Systems, International), as previously described (29). The
accuracy and precision of the system are tested monthly
using propane combustion tests. The average O2 and CO2
recoveries during the course of this study were 98.0%. Total
EE and substrate oxidation were calculated using oxygen
consumption and respiratory quotient (30). Urine N was
used to calculate 24-hour protein oxidation, with 1 gm of
urine N reflecting 6.25 gm of oxidized protein.
During the inpatient stay, participants wore an accelerometer (Actigraph GT1M, Pensacola, FL), which was
removed during exercise, sleep, and showering. EE from
activity was estimated from the accelerometer. If activity
counts were less than or equal to 1,952/min, the Work
Energy Theorem was used to estimate EE:
Kcal/minute = (0.0000191) (counts/minute)
(body mass [kg]).
11655
EB
EB CHANGES
CHANGES ANABOLIC
ANABOLIC EFFECT
EFFECT
Hypocaloric
Hypercaloric
CHO
Fat
Free Living
Lead in
Inpatient
Free Living
Lead in
Inpatient
Free living
Lead in
Inpatient
1.27 0.11
1.13 0.09
1.16 0.04
1.23 0.07
1.07 0.04
1.20 0.00
3.87 0.37
3.24 0.23
4.23 0.15
4.53 0.27*
3.91 0.14
5.87 0.36*#
1.18 0.11
1.13 0.14
1.03 0.04
1.10 0.06
0.95 0.03
1.35 0.07
EB
Energy Intake (kcal)
Free Living
Lead in
2,016 136
1,914 180
2,003 122
2,160 161
Inpatient
1,860 126
2,665 224*
Nonexercise PAL
CHO
PRO + CHO
CHO
PRO + CHO
CHO
PRO + CHO
1.60 0.02
1.59 0.04
1.61 0.02
1.57 0.04
1.36 0.02
1.37 0.02
1.37 0.03
1.35 0.03
285 35
360 51
291 40
354 88
Notes: bw = body weight; EB = energy balance; EE = energy expenditure; and PAL = physical activity level.
*Significant difference from free-living conditions within the same cohort.
#Significant difference from lead-in conditions within the same cohort.
Results
Hypocaloric
Hypercaloric
PAL
61166
MINOR ET AL.
0.94 to 2.05 gm N; p = .002; Figure 4C). There was no difference in mean NBAL between the CHO and PRO + CHO
trial in the negative EB group (CHO: 0.43 0.42 gm N,
1.39 to 0.53 gm N; PRO + CHO: 0.15 0.47 gm N,
1.24 to 0.94 gm N; p = .18; Figure 4D). However, mean
NBAL was greater (p = .016) in the PRO + CHO compared
with CHO in the positive EB group (CHO: 0.96 0.34 gm
N, 0.25 to 1.69 gm N; PRO + CHO: 1.49 0.26 gm N, 0.94
to 2.05 gm N; p = .016; Figure 4D).
Discussion
The current studies investigated the effects of the timing
of protein intake in relation to a bout of aerobic exercise on
NBAL in older individuals with varying energy intakes. Older
individuals completed two 3-day isocaloricisonitrogenous
trials with only the timing of protein consumption differing
between the two conditions. Contrary to our initial hypotheses, NBAL did not differ in older individuals on either
hypocaloric or hypercaloric diets when protein was consumed immediately after moderate aerobic exercise rather
than earlier in the day. However, further stratification of
positive versus negative EB to account for EB variations
within a trial revealed a strong influence of EB on the anabolic effect of postexercise feeding. To our knowledge, this
study is unique given its aim to investigate the combined
Figure 2. Mean daily body weight during inpatient period for both negative
energy balance (EB; n = 10) and positive EB (n = 6) groups (mean SEM).
EB
EB CHANGES
CHANGES ANABOLIC
ANABOLIC EFFECT
EFFECT
11677
Figure 4. Combined data from all three studies to determine the effect of energy balance (EB) on nitrogen balance. When combining all data without regard
to EB, NBAL was greater in PRO + CHO compared with CHO (p < .01; A). Restratified negative EB was lower compared with positive EB (p < .0001; B).
When restratified by treatment, positive EB had greater NBAL in both CHO (p = .009) and PRO + CHO groups (p = .002; C). When restratified by EB, there was no
increase with PRO + CHO in the negative EB group (p = .18), but there was an increase with PRO + CHO in the positive EB group (p = .016; D).
81168
MINOR ET AL.
EB
EB CHANGES
CHANGES ANABOLIC
ANABOLIC EFFECT
EFFECT
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