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Human Performance Laboratory, Ball State University, Muncie, Indiana 47306
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ABSTRACT |
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The purpose of this
investigation was to determine the effects of postexercise eucaloric
carbohydrate-protein feedings on muscle glycogen restoration after an
exhaustive cycle ergometer exercise bout. Seven male collegiate
cyclists [age = 25.6 ± 1.3 yr, height = 180.9 ± 3.2 cm, wt = 75.4 ± 4.0 kg, peak oxygen uptake (
O2 peak) = 4.20 ± 0.2 l/min] performed three trials, each separated by 1 wk:
1) 100%
-D-glucose [carbohydrate
(CHO)], 2) 70% carbohydrate-20% protein (PRO)-10% fat,
and 3) 86% carbohydrate-14% amino acid (AA). All feedings
were eucaloric, based on 1.0 g · kg body
wt
1 · h
1
of CHO, and administered every 30 min during a 4-h muscle glycogen restoration period in an 18% wt/vol solution. Muscle biopsies were
obtained immediately and 4 h after exercise. Blood samples were drawn
immediately after the exercise bout and every 0.5 h for 4 h during the
restoration period. Increases in muscle glycogen concentrations for the
three feedings (CHO, CHO-PRO, CHO-AA) were 118 mmol/kg dry
wt; however, no differences among the feedings were
apparent. The serum glucose and insulin responses did not differ
throughout the restoration period among the three feedings. These
results suggest that muscle glycogen restoration does not appear to be
enhanced with the addition of proteins or amino acids to an eucaloric
CHO feeding after exhaustive cycle exercise.
glucose; insulin; supplement; cycling
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INTRODUCTION |
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IT WAS FIRST REPORTED IN THE late 1960s that carbohydrate (CHO) ingestion after intense exercise promotes the restoration of muscle glycogen (2). Since then, a considerable amount of research has been conducted addressing the nutritional concerns of muscle and how various diets impact muscle metabolism and performance (cf. Ref. 6). From these data, it became evident that ingesting CHO before, during, and after exercise was beneficial to intense exercise performance.
More recently, attention has been directed toward consuming a combination of CHO and protein (PRO) after strenuous exercise to enhance muscle glycogen restoration (21, 23, 25). The ingestion of CHO-PRO complexes serves to increase insulin levels to a greater extent than CHO alone, thereby increasing glucose disposal into the muscle (25). However, these investigations have not standardized the caloric content of the feeding (25) or were unable to maintain an elevated insulin response throughout the restoration period (21, 23). Also, these investigations (21, 23, 25) did not examine the effects of eucaloric CHO and CHO-PRO feedings at rest. Thus it remains unclear if CHO-PRO feedings provide a greater insulinotrophic response (at rest and during recovery from exercise) and if these types of feedings are more beneficial for the restoration of muscle glycogen compared with the "classical" CHO feeding regimen after exhaustive exercise.
It was our intent to compare the effects of eucaloric CHO, CHO-PRO, and CHO-AA feedings on 1) muscle glycogen restoration after exhaustive exercise and 2) the glucose and insulin response at rest via an oral glucose tolerance test (OGTT) and during recovery from exhaustive exercise. We hypothesized that the CHO-PRO feedings would have a synergistic effect on the serum insulin response and result in a restoration of muscle glycogen above that produced with a CHO-only feeding.
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METHODS |
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Subjects
Eight male collegiate cyclists (nonsmokers with no metabolic or cardiovascular disorders) were recruited for this investigation. In addition, subjects were required to be glucose tolerant, having a fasted serum glucose concentration of <115 mg/dl, a peak value of <200 mg/dl, and a 2-h concentration of <140 mg/dl (16). One subject's peak serum glucose concentration was 214 mg/dl and the 2-h concentration was 156 mg/dl; therefore, this subject was dismissed from the investigation, and all data presented are based on seven subjects (Table 1). Subjects were informed of all experimental procedures and risks associated with the investigation and gave their written, informed consent in accordance with the University Institutional Review Board.
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Subjects performed three different muscle glycogen depletion/restoration trials, each separated by 1 wk. One trial investigated the effect of a CHO-only feeding on muscle glycogen restoration, whereas the other two trials consisted of a 70% CHO-20% PRO-10% fat feeding and an 86% CHO-14% amino acid (AA) feeding (see Experimental Feedings). All trials were performed in random order.
Preliminary Testing
Aerobic capacity test.
One week before the initial trial, subjects performed a graded exercise
test on an electronically braked cycle ergometer (Lode, Groningen,
Holland) to determine aerobic capacity or peak oxygen uptake
(
O2 peak). A one-way,
nonrebreathing respiratory valve and a nose clip were utilized to
collect expired oxygen and carbon dioxide. A personal computer was
interfaced with a Parkinson-Cowan dry-gas meter, an oxygen analyzer
(Applied Electrochemistry S-3A), and a carbon dioxide analyzer
(Sensormedics LB-2) to obtain the respiratory exchange ratio
and oxygen consumption (
O2)
values every 30 s.
O2 peak was
determined when subjects attained a respiratory exchange ratio value
>1.10 and/or a
O2
increase from the previous exercise intensity of <0.2 l/min (14, 24).
70% Check ride.
Two days after the
O2 peak test,
subjects underwent a 20-25 min
O2 check ride on a cycle
ergometer (Cybex, Ronkonkama, NY). Due to the differences in cycle
ergometers (Cybex vs. Lode) used for the
O2 peak test and the
muscle glycogen depletion rides, the check ride established the
appropriate exercise intensity necessary to achieve 70% of the
subject's predetermined
O2 peak for the
muscle glycogen depletion rides. This test also established the
appropriate workload necessary to elicit 125% of the subject's
O2 peak for the
sprints. During the check ride, the subject's
O2 was analyzed in a
process identical to that used in the
O2 peak test.
Experimental Trials
Each of the three muscle glycogen depletion/restoration trials was composed of a 4-day period. On days 1 and 2 of the trial, subjects consumed their "normal" diet. On day 3, subjects performed a fasted OGTT, consumed a controlled diet, and cycled in the evening for 45 min. On day 4, subjects cycled for 75 min and underwent six, 1-min sprints, followed by a 4-h muscle glycogen restoration period. This cycle ergometer protocol was modified from previous studies that demonstrated it to be an effective mechanism for decreasing muscle glycogen concentrations (15). An overview of the 4-day muscle glycogen depletion/restoration protocol is presented in Fig. 1.
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Dietary Control (Days 1, 2, and 3)
On days 1 and 2 of the trial, subjects consumed their normal dietary intake over the 48-h period. Diets were analyzed for total caloric intake and for amounts (g) of CHO, PRO, and fat by a dietary analysis program (ESHA Food Processor, version 7.11). A copy of this diet was given to the subjects to repeat for the ensuing two trials. This was done to assist in the standardization of muscle glycogen concentration before each trial. In addition, no exercise was permitted during this 48-h period.On day 3 of each trial, subjects were fed a controlled diet
(breakfast, lunch, and dinner) consisting of 55% CHO, 20% PRO, and
25% fat. The total caloric intake for each subject was based on the
Harris-Benedict (12) equation for men: total daily expenditure = {[66 + (13.7 × weight) + (5 × height)
(6.8 × age)] × activity factor × injury
factor}. All calories for the controlled diet on day 3 were consumed after the OGTT and before the 45-min ride that evening.
OGTT (Day 3: Morning)
On day 3 of the trial, after a 12-h overnight fast, subjects performed an OGTT. A 75-g (300 calories) load of
-D-glucose was dissolved in 400 ml of water and
administered for the OGTT of the CHO trial (16). The OGTT for the
CHO-PRO and CHO-AA trials were based on the 75-g load administered in
the CHO trial. The feeding consumed for the OGTT was identical to the
one used during the muscle glycogen restoration phase on day 4 of the trial. Before the OGTT, a fasted blood sample was collected
from an anticubital vein, via Teflon catheter kept patent with 0.9%
saline. After the fasted blood sample, subjects consumed the feeding,
and a blood sample was then taken every 30 min for 2 h (16).
Muscle Glycogen Depletion Protocol
Day 3: evening
The evening of day 3, subjects reported to the laboratory and
exercised on a cycle ergometer (Cybex) for 45 min at 70% of their
O2 peak. At 15 and 45 min, the subject's heart rate was measured via telemetry (Polar
Vantage XL, Polar Electro, Port Washington, NY), and
O2 was measured by
collection of expired air into Douglas bags for 60 s. Douglas bags were
analyzed for oxygen and carbon dioxide using gas analyzers to determine
O2. After the 45-min ride,
subjects were instructed to consume no additional food and, to ensure
adequate hydration, were given 1 liter of water to consume that evening.
Day 4.
On day 4 of the trial, subjects reported to the laboratory in
the morning and exercised for 75 min at 70% of their
O2 peak on a cycle
ergometer (Cybex). This was followed by six, 1-min sprints at 125% of
their
O2 peak with a
1-min rest period between sprints. Additional sprints were performed if
subjects were not completely exhausted. The subject's heart rate and
O2 were measured at 15, 45, and 75 min, via telemetry and Douglas bag collection, respectively.
Throughout the 45- and 75-min rides, subjects were allowed to consume
water ad libitum for the first trial. For the ensuing two
trials, individual water consumption was limited to the amount consumed
during the initial trial.
Muscle Biopsy
At the conclusion of the final sprint, a muscle biopsy was obtained with aid of suction (1, 9) from the vastus lateralis muscle. The time from cessation of exercise to the muscle biopsy was ~2 min. At the conclusion of the 4-h restoration period, another needle biopsy of the vastus lateralis muscles was obtained, ~3 cm proximal to the previous sample (8). The muscle samples were dissected of any visible connective tissue and immediately frozen in liquid nitrogen until assayed for total muscle glycogen.Blood Sampling
Before the 75-min muscle glycogen depletion ride was started, a fasted blood sample was obtained from an anticubital vein, via a vacutainer vial containing SST gel and clot activator (Vacutainer, Franklin Lakes, NJ). Blood samples were obtained immediately after the depletion ride and every 30 min thereafter for 4 h. All samples were centrifuged at 3,000 rpm for 15 min, and the serum was decanted from each sample and frozen at
10°C until analyzed for glucose and insulin.
Experimental Feedings
Consumption of the first feeding occurred immediately after the initial biopsy and blood sample. For the remainder of the restoration period, subjects consumed seven additional feedings, each separated by 30 min. The last feeding was consumed at the 210-min point, for a total of eight feedings. All feedings were eucaloric, based on 1.0 g · kg body wt
1 · h
1
of CHO, which has been shown to maximize muscle glycogen restoration (3, 14) and consisted of an 18% wt/vol solution, establishing similar
osmolalities within feedings. An example feeding for a 75-kg male is
presented in Table 2.
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The CHO feeding contained
-D-glucose. The CHO-PRO
feeding consisted of dextrose, PRO blend (caseinate, milk, and whey
protein), fructose, maltodextrine, and vegetable oil. The proteins
within the CHO-PRO feeding have previously demonstrated (17, 22, 25) to
elicit an elevated insulin response both with and without carbohydrates. The CHO-AA contained sucrose, fructose, dextrose, and
L-lysine, L-leucine, L-valine,
L-phenylalanine, L-threonine, L-histidine, L-isoleucine, and
L-methionine. It was hypothesized that, because exercise
stimulates the release of nonessential amino acids (5, 10), the CHO-AA
feeding containing only essential amino acids would produce an insulin
response greater than that demonstrated by the CHO-only feeding.
Muscle Glycogen Analysis
Muscle samples were sectioned into three pieces and subsequently freeze-dried (FTS Systems, Stone Ridge, NY) at
35°C for 7 days. Once the muscle sample was freeze-dried, each piece was weighed
on a microbalance (CAHN C-35 ATI Orion, Boston, MA) and placed in
hydrochloric acid for the hydrolysis of glycogen to glucose residue.
After hydrolysis, the sample was neutralized with sodium hydroxide, and
a reagent cocktail was added. The total muscle glycogen was determined
fluorometrically (Ratio-2 system fluorometer, Optical Technology
Devices, Elmsford, NY) by measuring the appearance of NADPH.
Blood Analysis
Serum glucose was determined by an enzymatic hexokinase/glucose-6-phosphate dehydrogenase reaction (Sigma Chemical no. 16-UV, St. Louis, MO). The absorbance of NADPH was measured on a spectrometer (spectrometer 501, Milton Roy) at 340 nm. A RIA kit (Coat-A-Count, Diagnostic Products, Los Angeles, CA) was used to analyze the serum insulin concentration of each sample in duplicate. Serum insulin samples, after a 24-h incubation, were analyzed via a gamma counter (1470 Wizard gamma counter, Wallac, Gaithersburg, MD). Each subject's glucose and insulin samples were assayed simultaneously to account for interassay variances.Statistical Analysis
Comparison of means between trials was conducted using a two-way repeated-measures ANOVA (treatment × time). Significance level was set at P < 0.05. When significance occurred between means, a Tukey's post hoc test was used to determine where significance occurred. All data are presented as means ± SE.| |
RESULTS |
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OGTT
Glucose.
The mean serum glucose concentration for the three trials increased
32% from baseline to 30 min (peak concentration), and, at the
conclusion of the OGTT, the response returned to baseline measurements
(Fig. 2). The glucose concentrations at 0, 30, 90, and 120 min were similar among the three trials. However, at 60 min, the serum glucose concentration for the CHO trial was greater (P < 0.05) than that of the CHO-PRO and CHO-AA trials. No
statistical differences were observed for the area under the curve
(AUC) in the CHO (150.1 ± 18.6 mM), CHO-PRO (96.1 ± 12.3 mM), and
CHO-AA (97.0 ± 18.6 mM) trials.
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Insulin. During the 2-h OGTT, no differences were observed at any point for the serum insulin concentrations (Fig. 2). The average insulin concentration for the three trials peaked at 30 min, and, at the conclusion of the OGTT, the insulin response returned to the baseline measurements. Additionally, no differences were observed for the AUC for CHO (2,671.2 ± 429.1 µU/ml), CHO-PRO (2,413.1 ± 316.7 µU/ml), and CHO-AA (1,964.3 ± 338.3 µU/ml) trials.
Glucose and Insulin Response During the 4-h Restoration Period
Glucose.
Serum glucose concentrations increased, on average, from 4.38 ± 0.08 mM before exhaustive exercise to 5.28 ± 0.28 mM immediately postexercise for the three trials (Fig. 3).
With serial feedings administered throughout the restoration period,
the mean serum glucose concentration peaked at 60 min, with an increase
of 33% from the immediate postexercise glucose concentration. There
was a 12% decrease in the serum glucose concentration from 60 to 120 min. From 120 min to the conclusion of the 4-h restoration period, the
mean serum glucose concentration ranged from 5.00-6.11 mM for all
three trials. However, the CHO trial at 90 min had a greater (P < 0.05) glucose concentration than either the CHO-PRO and CHO-AA trials. In addition, the AUC did not differ for the three trials (CHO = 287.9 ± 35.0, CHO-PRO = 277.8 ± 14.4, and CHO-AA = 211.6 ± 37.6 mM).
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Insulin. Throughout the restoration period, no differences were observed in the serum insulin concentrations at any time (Fig. 3). The insulin response of the three trials averaged an increase of 87% from immediately postexercise to the 120-min mark of the restoration period and remained elevated until the conclusion of the restoration period. At no time during the restoration period did the insulin concentrations approach the baseline concentrations found immediately postexercise; this can be attributed to the serial feedings administered every 0.5 h. The AUC during the restoration period did not differ for the three trials (CHO = 5,095.7 ± 1,231.9, CHO-PRO = 4,567.3 ± 843.7, and CHO-AA = 3,216.0 ± 630.9 µU/ml).
Muscle Glycogen Restoration
The muscle glycogen concentrations immediately after exercise were similar among the three trials (Fig. 4) (CHO = 107 ± 27, CHO-PRO = 118 ± 35, and CHO-AA = 87 ± 36 mmol/kg dry wt). As expected, there was an increase (P < 0.05) in muscle glycogen concentrations at the conclusion of the 4-h restoration period (CHO = 231 ± 37, CHO-PRO = 230 ± 29, and CHO-AA = 205 ± 40 mmol/kg dry wt); however, no differences were apparent among the three trials. Over the 4-h restoration period, the three feedings caused an average increase in muscle glycogen concentration of 118 mmol/kg dry wt.
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Dietary Intake
On days 1, 2, and 3 of each trial, no differences were observed in the amount of calories, CHO, PRO, and fat (grams and percent) consumed (Table 3). Because the dietary consumption for the initial 3 days was identical for all trials, no differences were observed among the trials.
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Depletion Ride and Sprints
The
O2 on days 3 and
4 during the 45- and 75-min rides were similar (70.4 ± 4.4%
and 70.9 ± 5.0% of
O2 peak,
respectively), and no difference were observed among trials. The number
of sprints (CHO = 7.4 ± 2.6, CHO-PRO = 7.6 ± 1.6, and CHO-AA = 7.4 ± 2.7) per trial and the watt output for the sprints (CHO = 435 ± 41, CHO-PRO = 435 ± 38, and CHO-AA = 439 ± 38 W) were similar
within and among the three trials.
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DISCUSSION |
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The purpose of this investigation was to examine if different CHO-PRO feedings would enhance muscle glycogen restoration after an exhaustive exercise bout. More specifically, muscle glycogen restoration was examined using serial eucaloric CHO, CHO-PRO, and CHO-AA feedings over a 4-h restoration period after exhaustive cycle ergometer exercise. The primary finding of this investigation was that the addition of protein or amino acids to an eucaloric postexercise CHO feeding did not appear to enhance the restoration of muscle glycogen compared with a CHO-only feeding.
In the present study, the three eucaloric feedings that were
administered were based on 1.0 g · kg body
wt
1 · h
1
of CHO, with no differences observed in muscle glycogen restoration among the three trials. These results contradict the findings of
Zawadzki et al. (25), in which subjects were administered three
feedings: 1) 112 g of CHO, 2) 40.7 g of PRO, and
3) 152.7 g of a CHO-PRO mixture. They demonstrated that, during
a 4-h restoration period, the CHO-PRO feeding elicited a 38% greater
muscle glycogen restoration than the CHO-only feeding, and both were
greater than the PRO-only feeding.
Two previous investigations (21, 23) similar to the present study examined the effects of isoenergetic CHO and CHO-PRO feedings (based on 1.0 g/kg body wt of CHO) on muscle glycogen restoration after an exhaustive exercise bout. These results demonstrated that both feedings had similar effects on muscle glycogen restoration. The results from the present investigations, along with those of Roy and Tarnopolsky (21) and Tarnopolsky et al. (23), demonstrate that the caloric content of the feedings may impact muscle glycogen restoration. The additional caloric content of the CHO-PRO feeding in the investigation by Zawadzki et al. (25) may have been a critical determinate for the enhanced muscle glycogen storage of the CHO-PRO feeding compared with the CHO- and PRO-only feedings.
The muscle glycogen concentration immediately after the exhaustive exercise bout in the present investigation averaged 104 mmol/kg dry wt for the three trials. These findings are similar to the results reported by Tarnopolsky et al. (23), who also used endurance trained cyclists. In contrast, the investigation by Roy and Tarnopolsky (21) reported that the immediate postexercise muscle glycogen concentrations for the CHO and CHO-PRO trials were ~228 mmol/kg dry wt. The most likely cause for the differences in muscle glycogen concentrations between the studies is the different exhaustive exercise protocols, i.e., resistance training in the investigation by Roy and Tarnopolsky (21) vs. a cycle ergometer protocol in the present investigation and that of Tarnopolsky et al. (23). At the conclusion of the 4-h restoration period, a greater absolute increase in muscle glycogen concentration (118 vs. 84 mmol/kg dry wt) was evident in the present investigation compared with the increase found by Roy and Tarnopolsky (21), which could result in an elevated glycogen synthase activity (13, 19, 24).
Previous investigations by Blom et al. (3) and Ivy et al. (14)
demonstrated that CHO ingestion between 0.70 and 1.5 g · kg body
wt
1 · h
1
after exhaustive exercise provided adequate stimulus for maximal muscle
glycogen restoration over a 4-h period. However, additional CHO
ingestion (>0.7 g · kg body
wt
1 · h
1)
did not appear to provide an enhanced benefit in the restoration of
muscle glycogen (3, 14). The CHO content of the three feedings in the
present investigation differed (CHO = 1.0, CHO-PRO = 0.71, and CHO-AA = 0.86 g · kg body
wt
1 · h
1),
but muscle glycogen restoration among the three trials was similar, a
finding that supports the results of Blom et al. (3) and Ivy et al.
(14). Therefore, muscle glycogen restoration does not appear to be
altered with a CHO-PRO feeding, provided that adequate carbohydrates
are consumed.
The addition of protein to a CHO feeding has been shown to have a synergistic effect on the serum insulin response compared with a CHO-only feeding (17, 18, 22, 25). Zawadzki et al. (25) theorized that the elevated plasma insulin response in the CHO-PRO trial was responsible for the decrease in the serum glucose concentrations and for the greater muscle glycogen restoration compared with the CHO and PRO feedings. The addition of protein or amino acids to the CHO feeding in the present study and in previous investigations (21, 23) did not elicit a synergistic insulin response. Furthermore, in the present study, during the resting OGTT and restoration periods, no significant differences were found in the insulin and glucose responses for the three feedings. Likewise, the AUC for the glucose and insulin responses demonstrated similar responses for the three trials during both the resting OGTT and the 4-h restoration period. Neither the investigations by Roy and Tarnopolsky (21) and Tarnopolsky et al. (23) nor the present study demonstrated greater muscle glycogen restoration with CHO-PRO or CHO-AA feedings compared with CHO-only feeding, thus indicating that the caloric intake of the feedings does impact serum insulin concentrations. Lastly, it should be noted that the type of proteins used for the feedings does not appear to be identical among the studies and may have contributed to the differences observed in blood insulin and muscle glycogen concentrations.
The serum glucose responses at 60 min of the resting OGTT and at 90 min
of the 4-h restoration period in the present study were greater in the
CHO trial than in either the CHO-PRO or the CHO-AA trial. The elevated
glucose response may be due to the greater CHO load in the CHO trial
compared with the CHO-PRO and CHO-AA trials (1.0 vs. 0.70 and 0.86 g · kg body
wt
1 · h
1,
respectively). Additionally, after the 60- and 90-min time points of
the resting OGTT and 4-h restoration period, respectively, the serum
insulin response demonstrated a trend to be greater during the CHO
trial than in the CHO-PRO and CHO-AA trials, thereby compensating for
the elevated glucose response elicited at the 60- and 90-min time points.
Carbohydrate metabolism may be altered by the simultaneous ingestion of fats, as each substrate competes as an oxidative fuel source (20). Therefore, the addition of fat in the CHO-PRO feeding may have decreased glucose utilization, and a concomitant increase in the serum glucose concentration would be expected. Previous investigations (4, 21, 23) have demonstrated that the addition of minimal amounts of fats (11 and 31%) to a CHO-only feeding did not effect the glucose response or muscle glycogen restoration. The CHO-PRO feeding demonstrated a serum glucose response simliar to that for CHO-AA and CHO feedings in the present study, supporting the findings of previous investigations (4, 21, 23).
In summary, muscle glycogen concentrations for the three trials were
similar immediately postexercise and again 4 h after the cessation of
exercise. In addition, the serum insulin and glucose responses among
the three eucaloric feedings displayed no differences at any time
throughout the 4-h restoration period. Therefore, it appears that the
addition of protein or amino acids to an eucaloric postexercise CHO
feeding does not enhance the restoration of muscle glycogen compared
with a CHO-only feeding. Thus, provided the caloric content is similar
and adequate amounts of CHO are consumed (>0.70
g · kg body
wt
1 · h
1)
after exhaustive exercise, the addition of protein or amino acids to a
CHO feeding does not appear to enhance muscle glycogen restoration.
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ACKNOWLEDGEMENTS |
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We thank Bill Fink for technical assistance in analysis of blood and muscle samples.
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FOOTNOTES |
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This project was supported, in part, by a grant from General Nutrition, Pittsburgh, PA.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. W. Trappe, Human Performance Laboratory, Ball State Univ., Muncie, Indiana 47306 (E-mail: strappe{at}bsu.edu).
Received 27 June 1999; accepted in final form 26 January 2000.
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