Journal of Exercise Physiologyonline

October 2014Volume 17 Number 5

Editor-in-ChiefTommy Boone, PhD, MBAReview BoardTodd Astorino, PhDJulien Baker, PhDSteve Brock, PhDLance Dalleck, PhDEric Goulet, PhDRobert Gotshall, PhDAlexander Hutchison, PhDM. Knight-Maloney, PhDLen Kravitz, PhDJames Laskin, PhDYit Aun Lim, PhDLonnie Lowery, PhDDerek Marks, PhDCristine Mermier, PhDRobert Robergs, PhDChantal Vella, PhDDale Wagner, PhDFrank Wyatt, PhDBen Zhou, PhD

Official Research Journal of the American Society of

Exercise Physiologists

ISSN 1097-9751

Official Research Journal of the American

Society of Exercise Physiologists

ISSN 1097-9751

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JEPonline

The Addition of Protein to a Carbohydrate Endurance Supplement Does Not Enhance Running Performance

Craig O. Mattern1, Brian Campbell2, Tina Carson1, Justin Charland2, William S. Craven3, Natalia Filip2, Celia Watt1, Ryan Yaple1, Heidi K. Byrne1

1The College at Brockport, SUNY, Brockport, NY, 2Upstate Medical University, SUNY, Syracuse, NY, 3Ball State University, Muncie, IN

ABSTRACT

Mattern CO, Campbell B, Carson T, Charland J, Craven WS, Filip N, Watt CA, Yaple R, Byrne HK. The Addition of Protein to a Carbohydrate Endurance Supplement Does Not Enhance Running Performance. JEPonline 2014;17(5):31-42. It is unclear whether or not the isocaloric addition of protein (PRO) to a carbohydrate (CHO) endurance exercise supplement improves exercise performance and/or recovery. Thus, the two-fold purpose of this study was to: (a) determine if a PRO+CHO beverage ingested during and/or after endurance exercise improves performance in a subsequent exercise bout compared to CHO alone; and (b) if the timing of the supplement influences recovery and subsequent exercise performance. Using a randomized crossover design, 9 endurance trained subjects (age = 33 ± 12.7 yrs; VO2 max = 65.1 ± mL·kg-1·min-1, body fat = 7.9 ± 2.98%) received a beverage containing CHO (0.65 g of CHO·kg-1) or CHO + PRO (0.52 g of CHO·kg-1 plus 0.13 g of protein·kg-1) participated in a 1-hr run at 67% ± 5.4 of VO2 max. During a 7-hr recovery period, the subjects were given beverages that contained either CHO (1.0 g of CHO·kg-1) or CHO+PRO (0.80 g of CHO·kg-1

plus 0.20 g of protein·kg-1) immediately post-exercise and at 1 hr and 4 hrs of during recovery. Then, the subjects ran a 10-km time trial. There were no statistical differences in blood glucose or insulin during or after exercise among the four nutritional conditions. There was no difference between type or timing of the supplement on the 10-km performance, suggesting that the combination of PRO+CHO affords no additional benefit when compared to CHO alone.

Key Words: Exercise, Recovery, Supplement, Time Trial Run

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INTRODUCTION

Nutrition has been determined to be a key element in the recovery from strenuous physical activity (6). Historically, most liquid based endurance exercise supplements have been comprised of carbohydrate as well as various combinations of electrolytes, vitamins, and minerals. More recently however, a variety of manufacturers have begun to market supplements containing a combination of both carbohydrate and protein, often in a 4:1 ratio. Manufacturers purport that the inclusion of protein in a recovery beverage may provide the added benefit of improved exercise performance, recovery, fluid retention, and reduced muscle damage. Yet, the handful of scientific investigations of these claims has yielded varied outcomes. Some investigators have demonstrated the addition of protein improved performance and recovery (7,9,16,17) while other researchers (3,12,14,21) have observed no differences compared to a supplement containing only carbohydrate.

It is likely that two major variables account for much of the divergence in this body of literature. One of these factors is whether the authors employed an isocaloric design. In other words, does the study design add protein to a carbohydrate beverage or replace some of the carbohydrate in the beverage with protein. Some studies (7,9,16,18) that added protein to existing carbohydrate content found an improvement in performance. But, the question remains as to whether the improvement was due to the protein or simply the increased caloric content. Many studies (3,12,14) that employ an isocaloric design show no improved performance.

The second notable factor that plays a role in this body of literature is likely the timeframe in which the supplement is provided relative to exercise. Some investigators (7,18) provide supplementation during exercise while others (3,9,14) use it exclusively post-exercise. Both investigations which provide supplementation during exercise demonstrated an enhanced time to exhaustion in the carbohydrate plus protein condition, but the generalizability of these data are limited in that neither of these investigations employed an isocaloric design. Of the investigations providing supplementation post-exercise, the only study (9) to show a performance benefit did not use an isocaloric design. Both isocalorically designed post-exercise supplementation investigations demonstrated no performance benefit in the carbohydrate plus protein condition (3,14). It should be pointed out that none of these investigations were designed to study the optimal timing of supplement provision. Hence it is unclear as to whether the timing of delivery of a protein containing beverage influences its effectiveness of enhancing recovery.

Therefore, the aims of this investigation were to: (a) compare the effectiveness of an isocaloric supplement containing either carbohydrate alone (CHO+CHO) or a combination of carbohydrate and protein (CHO+PRO); and (b) determine if the timing (during vs. post-exercise) of the supplement provision influences recovery and subsequent exercise performance.

METHODS

Subjects Nine endurance trained male subjects volunteered to participate in this double-blind investigation which employed a randomized cross-over design (see Table 1 for descriptive characteristics). All subjects had physician clearance prior to participation. None had uncontrolled hypertension, heart arrhythmia, and lung disease. Each subject had aerobically trained a minimum of 8 hrs·wk -1 and/or participated in two or more organized running races 6 months prior to enrolling in the investigation.

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Table 1. Subject Characteristics.

  Characteristic Mean ± SD (N = 9)

Age (yr) 33.3 ± 12.67

Height (cm) 180.0 ± 4.81

Weight (kg) 72.8 ± 5.77

VO2 max (mL·kg-1·min-1) 65.1 ± 4.42

Body fat (%) 7.9 ± 2.98

Procedures Baseline Measurements The subjects’ body weight was measured using a digital scale. Height was measured with a stadiometer. Body density was estimated using skinfold measures described by Jackson and Pollock (8). All measurements were performed by the same experienced technician using Harpenden calipers. The procedures described by Lohman et al. (10) were followed to estimate body density. Percent body fat was calculated using the Siri equation (20).

A VO2 max test was performed on a treadmill and was used to determine the intensity of the aerobic exercise for the subsequent four visits to the laboratory. The VO2 max test was conducted using a speed the subjects would typically run for 30 min. The test began at this speed with 0% grade. The speed remained constant throughout the test. The grade was increased by 2% every 2 min. Heart rate (HR) and gas exchange data were monitored continuously. Blood pressure was measured every 3 min. A Parvomedics TrueMax 2400 (Salt Lake City, Utah) was used for metabolic measurements.

Fatigue ProtocolThe subjects arrived to the laboratory for each of four visits at ~7:00 a.m. after having fasted for 10 hrs. Each subject: (a) recorded his diet for 2 days prior to each testing session; (b) consumed at least 500 mL of water the night prior to the test; (c) instructed not to perform vigorous exercise 18 hrs prior to each testing session; and (d) maintained his training status while enrolled in the investigation. Each of the 4 exercise trials was separated by ~7 days.

Upon arrival to the laboratory, the subjects were weighed and provided with a HR monitor. Each subject was then allowed to warm up for 5 min by running on the treadmill at 60% of his VO 2 max. The subjects were then instructed to run at 70% of their VO2 max for 60 min. The initial speed of the treadmill corresponded to 70% of the subjects’ VO2 max, which was determined using the following equation: VO2 mL·kg-1·min-1 = (0.2 x speed) + 3.5 mL·kg-1·min-1. During the first 5 min of the first experimental trial, VO2 was monitored and speed was adjusted if necessary such that the subject

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was exercising as close to 70% of VO2 max as possible. This speed was then replicated for the three subsequent experimental trials.

Heart rate was recorded at minutes 30 and 55 of the 1 hr run. Rating of perceived exertion (RPE) was recorded at minutes 10, 25, 40, and 55. Upon completion of the exercise, the subjects were allowed to cool down for 5 min and the recovery process began.

Recovery ProtocolThe recovery phase lasted a total of 7 hrs of which the subjects were allowed to leave the lab and go about their normal daily activities, but they were instructed not to exercise. During this time period, the subjects were asked not to eat or drink anything other than the recovery product that was provided.

Supplementation Composition and ProvisionThe two supplements used in this study were isocaloric solutions made up of either carbohydrate alone (CHO+CHO) or a combination of carbohydrate and protein (CHO+PRO). The supplements were designed by food technologists such that the flavor, texture, and mineral content of the two beverages were uniform. The CHO+CHO supplement was comprised of a mixture of maltodextrin, sucrose, and dextrose. The CHO+PRO supplement was made using a 4:1 ratio of CHO to PRO. The CHO portion was again made with a mixture of maltodextrin, sucrose, and dextrose and the PRO portion was comprised of a whey protein isolate. Both supplements contained 3.64 mg·kg -1 of sodium, 0.77 mg·kg-1 of potassium, 1.12 mg·kg-1 of calcium, and 1.47 mg·kg-1 of magnesium.

During the fatigue protocol the subjects consumed a total of 12 mL·kg -1 of supplement. The CHO+CHO supplement contained 0.65 g CHO·kg-1 and the CHO+PRO supplement contained 0.52 g CHO·kg-1 and 0.13 g PRO·kg-1 (see Table 2). The total volume of fluid was divided into four 3 mL·kg-1 aliquots. The subjects consumed each aliquot at minutes 10-15, 25-30, 40-45, and 55-60.

In order to provide adequate nutrition during the 7-hr recovery time span, the subjects consumed 3 servings of a more concentrated solution containing isocaloric supplements of either CHO+CHO (1.0 g CHO·kg-1) or CHO+PRO (0.80 g CHO·kg-1 and 0.20 g PRO·kg-1) (see Table 2). Each serving contained 12 mL·kg-1 of fluid. The first serving was consumed immediately after the cool down from the fatiguing bout of exercise. The subjects then departed the laboratory with two servings of pre-mixed supplement. The first was consumed 1 hr into recovery and the second was consumed 4 hrs into recovery. This allowed for a 3-hr digestion time between the consumption of the last serving and the beginning of the 10 km performance evaluation run.

Table 2. Fatigue and Recovery Supplement Protocol. Conditions Fatigue Protocol Supplement Recovery Supplement

A CHO + CHO (0.65 g CHO·kg-1) CHO + PRO (0.80 g CHO·kg-1+ 0.20 g PRO·kg-1)

B CHO + PRO (0.52 g CHO·kg-1+ 0.13 g PRO·kg-1) CHO + CHO (1.0 g CHO·kg-1)

C CHO + PRO (0.52 g CHO·kg-1+ 0.13 g PRO·kg-1) CHO + PRO (0.80 g CHO·kg-1+ 0.20 g PRO·kg-1)

D CHO + CHO (0.65 g CHO·kg-1) CHO + CHO (1.0 g CHO·kg-1)

CHO = carbohydratePRO = protein

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Performance ProtocolThe subjects returned to the laboratory after their 7-hr recovery (~3:30 p.m.) for the completion of a 10-km time trial performed on a treadmill. Upon arrival the subjects were again weighed and provided with a HR monitor. Following a 5 min warm up of running on the treadmill at 60% of their VO2 max, the subjects instructed to complete the 10-km distance in the fastest time possible. Each subject controlled the speed of the treadmill and could see the distance covered, but time and running speed displays were not visible to the subject. The subjects were allowed to drink as much water as they wanted during the run and the same amount was replicated for each of the three subsequent visits.

Blood Borne MeasurementsDuring the fatigue protocol, blood samples (~1 mL) were obtained from a finger-stick using a sterile technique at the following time intervals: baseline, 30 min into the fatiguing bout of exercise, and upon completion. During the recovery phase, each subject returned briefly (5 min) to the laboratory for a recovery finger stick blood sample at 2 and 4 hrs. Just prior to and upon completion of the performance trial, finger stick blood samples were obtained. All samples were used for the determination of glucose and insulin levels. Figure 1 presents the methodological timeline.

Figure 1: Methodological Timeline

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Blood Analysis Serum insulin concentration was measured via the enzyme-linked immunosorbent assay (ELISA) technique using DSL-10-1600 insulin ELISA kit (Diagnostic Systems Laboratories, Webster, TX). Analyses were performed in duplicate using an ELx800 Microplate Absorbance Reader, and an ELx50 Strip Washer (BioTek Instruments, Winooski, VT). Analysis of blood glucose was conducted using the Accu-Check Blood Glucose Monitoring System (Roche Diagnostics, Indianapolis, IN).

Statistical AnalysesAll statistical analyses were conducted using the SPSS software package (SPSS Inc., Chicago, IL). Descriptive statistics were calculated and variables were examined for meeting assumptions of normal distributions. All data are presented as means ± standard deviation (SD). General linear model (GLM) repeated measures analyses were used to test differences across the four nutritional trials at the various time points within each nutritional condition. A value of P≤0.05 level of significance was used in all analyses.

RESULTS

The subjects recorded their nutritional consumption for 2 days prior to each of the four experimental trials. The data were input into nutritional software (Nutritionist IV, Stafford, TX) for the determination of daily caloric content and macronutrient profiles. As demonstrated in Table 3, there were no statistical differences in average daily caloric intake among the four nutritional conditions. Additionally, the composition of these diets was quite similar in that no differences existed among the four nutritional conditions for percentages of carbohydrate, fat, or protein.

Table 3. Nutritional Intake Prior to Experimental Trials.

Measures A

Conditions B

C

D

Caloric intake (kcal·day-1) 3,012.0 ± 1,038.9 3,410.4 ± 1,090.2 3,029.2 ± 1,145.4 3,181.8 ± 751.5

Carbohydrate (%) 54.0 ± 7.8 51.4 ± 7.2 56.4 ± 6.2 56.4 ± 6.9

Protein (%) 17.5 ± 4.8 16.8 ± 1.8 16.4 ± 2.7 15.2 ± 3.0

Fat (%) 28.7 ± 7.5 29.8 ± 7.8 25.8 ± 8.4 27.0 ± 5.9

Data are presented as mean ± SD

The experimental design called for the subjects to perform the fatiguing bout of exercise at 70% of VO2 max. Oxygen consumption was monitored during the first 5 min of the first experimental trial. It was determined that subjects ran at 67 ± 5.4% of their VO2 max. Average HR during the fatiguing bout of exercise was 82.6 ± 4.86% of maximal HR. Average RPE during this bout of exercise was 12.9 ± 1.76.

Blood glucose levels were determined during the fatiguing bout of exercise, recovery, as well as before and after the 10-km performance run. Average blood glucose prior to exercise was 94.9 ± 0.8 mg·dl-1. In all nutritional conditions, values increased significantly (P<0.05) during exercise and

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decreased back toward baseline during recovery, only to significantly (P<0.05) increase again upon the completion of the 10-km performance run. However, at any given measurement time-point, there were no significant differences among the four nutritional conditions (refer to Figure 2).

Figure 2: Blood Glucose Responses during a 1-hr Fatigue Inducing Run, Recovery, and Before and After a 10-km Running Time Trial. There were no significant differences within any time point among the four nutritional conditions. Data are presented as mean ± SD.

Serum insulin levels were also determined during the fatiguing bout of exercise, recovery, as well as before and after the 10-km performance run. In all nutritional conditions, insulin levels remained stable during the initial phase of the fatiguing bout of exercise. Upon completion of the exercise bout, insulin levels tended to rise in the CHO+PRO fatigue ex/CHO+CHO recovery condition (P = 0.020), the CHO+PRO fatigue ex/CHO+PRO recovery condition (P = 0.078), and the CHO+CHO fatigue ex/CHO+CHO recovery condition (P = 0.029). These elevations were maintained through 2 hrs of recovery, and then the values regressed back towards baseline for the remainder of recovery and through the completion of the 10-km performance run. However within any time-point, there were no significant differences among the four nutritional conditions (Figure 3).

The average finish times for the 10-km running time trial were extremely similar among the four nutritional conditions. As demonstrated in Table 3, there were no statistical differences in finish times among the four conditions.

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Figure 3: Serum Insulin Responses during a 1-hr Fatigue Inducing Run, Recovery, and Before and After a 10-km Running Time Trial. There were no significant differences within any time point among the four nutritional conditions. Data are presented as mean ± SD.

Table 3. Performance Run Times for 10-km Time Trial.

A B C D

Run Times for10-km TT (min) 42.9 ± 4.73 42.4 ± 4.65 42.7 ± 4.92 42.8 ± 4.62

Data are presented as mean ± SD

DISCUSSION

This investigation was designed to study whether the isocaloric replacement of carbohydrate with protein would enhance exercise performance after a previous bout of fatigue inducing exercise. Additionally, we studied whether the timing of the supplement provision influenced subsequent exercise performance. In brief, the addition of protein provided during the bout of fatiguing exercise, during recovery or during both of these time frames did not increase subsequent exercise performance measured by a 10-km running time trial.

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Previous investigations on this topic have led to mixed findings. While some isocalorically designed experiments show enhanced performance after the ingestion of CHO+PRO during recovery (1,2,11,14), others have found no benefit to the replacement of CHO with PRO (3,12-14).

Algahannam (1) performed an investigation designed to evaluate appropriate supplementation to be consumed during a simulated European football match. In this study, the subjects were provided with either an isocaloric CHO, CHO + PRO or a placebo 15 min prior to and at halftime of the simulated football match. A run to fatigue at 80% VO2 max followed the game simulation. Run time to fatigue was significantly longer in the subjects after the consumption of CHO+PRO when compared to both CHO and placebo. While the design of this study can be applied directly to the nutritional demands of a sporting event, the absence of a recovery phase makes comparisons between this paper and our study (which used a 7-hr recovery period) very difficult. On the other hand, an investigation by Berardi et al. (2) used a 6-hr recovery phase, which was very similar to our recovery timeline. In this investigation, trained cyclists completed a 1-hr time trial. The subjects then consumed either a CHO or CHO+PRO supplement at hour 0, 1, and 2 of recovery. At hour 4 of the 6-hr recovery period, the subjects consumed a standardized meal. This was followed by a second 1-hr time trial. Unlike our investigation, Berardi et al. (2) found that time trial performance in the CHO + PRO condition was significantly better than that of the CHO condition. One plausible explanation for the divergent results between the Berardi et al. (2) study and this investigation may be the ratio of CHO:PRO utilized. We chose a 4:1 CHO:PRO ratio, as it is commonly available and often consumed by endurance athletes, whereas Berardi and colleagues (2) chose a 2:1 ratio. A second difference between the two investigations is the subjects’ familiarization to the performance measures. We did not familiarize our subjects to the 10-km running time trial effort while Berardi et al. (2) had their subjects perform two familiarization trials prior to testing. In a recent investigation, Lunn et al. (11) fatigued male runners using a 45-min run at 65% of VO 2

max. One dose of CHO+PRO or CHO supplementation was provided immediately following the exercise bout. The subjects recovered for 3 hrs and then performed an intense anaerobic time to exhaustion effort lasting approximately 4 min. Time to exhaustion performance was improved by 23% in the CHO+PRO condition versus the CHO condition. Although Lunn et al. (11) showed enhanced performance, the measure of performance, being an extremely intense and short duration exercise bout, is notably different from our 10-km running time trial which took the subjects approximately 43 min to complete. In addition, because chocolate milk was the CHO + PRO supplement, while a grape flavored beverage was the CHO control, a double blind design was not utilized, creating a potential for subject bias.

Rowlands et al. (14) designed an experiment to follow the recovery of the subjects over a 4-day span of time. On Day 1, trained cyclists performed 2.5 hrs of intervals designed to deplete muscle glycogen. The subjects were fed either a CHO+PRO or CHO recovery product at 3 time points during a 4-hr recovery. On Day 2, the subjects performed a sprint performance evaluation, after which they followed the same recovery protocol used on Day 1. On Day 3, the subjects consumed a standardized high CHO diet and did not exercise, and on Day 4 they repeated the sprint performance evaluation. There were no differences in the Day 2 mean sprint power output between the CHO versus the CHO+PRO conditions. These findings parallel our data. However, on Day 4 the mean sprint power was improved in the CHO + PRO compared to the CHO condition. While this improved performance is in contrast to our results, we employed a 7-hr recovery phase and it took 60 hrs of recovery for Rowlands et al. (14) to detect a performance benefit of the CHO+PRO supplement.

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This body of literature is mixed and several experiments directly support our findings indicating no performance advantage when using CHO+PRO versus CHO alone. Betts et al. (3) fatigued their subjects by having them run for 90 min at 70% of VO2 max. During 4 hrs of recovery, the subjects were provided with either 0.8 g of CHO·kg-1·hr-1 + 0.3 g of protein·kg-1·hr-1 or 1.1 g of CHO·kg-1·hr-1. Then, they performed a running time to exhaustion test in which there were no differences between nutritional conditions. We provided supplementation both during the fatiguing bout of exercise and during recovery, which would, in theory, potentiate any possible benefits that protein might offer. In addition we chose the outcome variable of a 10-km running performance time as opposed to time to exhaustion. However, the outcome of our investigation was the same as Betts et al. (3) in that we found no benefit to the isocaloric inclusion of protein into the supplement. Rowlands et al. (14) also found no clear benefit of an isocaloric protein-enriched recovery feeding. Ten endurance trained athletes performed 2.5 hrs of cycling intervals, followed by a 4-hr recovery period. During the recovery, the subjects were provided with either a high carbohydrate or a high protein supplement every 30 min. After an overnight fast the subjects then returned to the laboratory to perform a repeated maximal sprint performance test. The main difference in experimental design between the Rowlands et al. (14) investigation and the present study was the timing of the supplementation provision. However, the outcomes of the two investigations were very similar in that there were no differences in glucose, insulin, or subsequent exercise performance between nutritional conditions.

Romano-Ely et al. (12) also found no differences in time to fatigue when comparing an isocaloric CHO supplement versus CHO/PRO supplement. Their investigation required the subjects to cycle to exhaustion at 70% of VO2 peak while providing supplementation every 15 min. The subjects were provided supplementation only during the first 15 min of a 24-hr recovery. For the remainder of the recovery, they consumed their normal diet. The subjects returned to the laboratory the following day and rode at 80% of VO2 peak until exhaustion. Time to fatigue was not different between nutritional conditions for the 70% or 80% VO2 peak trials.

A more recent investigation by Richardson et al. (13) had moderately trained subjects exercise to exhaustion at 75% VO2 max. This was followed by a 3-hr recovery period in which the subjects were fed every 30 min for the first 2 hrs. The randomly assigned feedings were comprised of either a CHO (1.5 g·kg-1·hr-1) or a CHO + PRO (1.2 g·kg-1·hr-1 CHO + 0.3 g·kg-1·hr-1PRO). The subjects then performed a second time to exhaustion trial after the 3-hr recovery. There were no differences in the post-recovery time to exhaustion trial between the two nutritional conditions.

The Betts et al. (3), Rowlands et al. (14), and Richardson et al. (13) studies provided supplement only during recovery and not during the fatiguing exercise bout. On the other hand, Romano-Ely et al. (12) provided supplement during the fatiguing exercise bout and only at the 15-min time point of recovery. Due to these methodological discrepancies, we decided to take the novel approach of not only studying the potential performance benefit of protein, but also to determine if the time points (during exercise versus recovery) during which the protein was provided impacted its effectiveness (see Table 2). However, regardless of the manipulation of both composition and timing of the supplements, our investigation substantiates the findings of these previous investigations in that the isocaloric addition of PRO to a CHO supplement did not enhance post-recovery performance.

CONCLUSIONS

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The addition of protein provided during the bout of fatiguing exercise, during recovery, or during both of these time frames did not increase subsequent exercise performance measured by a 10-km running time trial. Therefore, these data suggest that endurance runners performing multiple bouts of exercise within one day do not need to consume CHO+PRO in a 4:1 ratio to enhance recovery and subsequent exercise performance.

ACKNOWLEDGMENTSThis project was supported by Infinit Nutrition.

Address for correspondence: Craig O. Mattern, PhD, Department of Kinesiology, Sports Studies and Physical Education, The College at Brockport, State University of New York, Brockport, NY, USA, 14420. Phone: (585) 395-5343; FAX: (585) 395-2771; Email: cmattern@brockport.edu

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