It has long been recognized that endurance exercise performance is significantly improved when carbohydrate is ingested during exercise compared to water-only or placebo beverages (5,6,14,34,35). Several recent investigations have reported significant improvements in endurance exercise performance when a carbohydrate-protein (CHO-PRO) beverage is ingested during exercise compared to a carbohydrate-only (CHO) beverage (15,27,28). The added amount of protein is typically 25-30% of the total energy content of the beverage, yet it appears to produce performance benefits beyond that of traditional carbohydrate-only beverages (3,15,25,27,28,33), and reduce muscle damage (2,27,28). However, many athletes and recreational exercisers desire a lower carbohydrate, lower caloric content alternative when maintaining or reducing body weight, which is a goal, in addition to improving fitness and endurance.
Previous work in our laboratory demonstrated that a lower CHO beverage with added protein was as effective in extending time to exhaustion (TTE) as higher carbohydrate and higher calorie beverages (24). Martinez-Lagunas et al. (24) recently compared the effects of a 4.5% CHO plus 1.15% PRO, and a 3% CHO plus 0.75% PRO beverage, to a traditional 6% CHO beverage and found that there was no difference in the times to exhaustion between the treatments. This suggests that the efficacy of the supplements was maintained despite the reduction in total CHO and total energy content with the substitution of a small amount of protein (24). Based on these findings, we sought to determine if a lower CHO, lower calorie beverage containing a moderate amount of protein could be optimized using a mixture of CHO sources (glucose [dextrose], maltodextrin, and fructose) rather than a single CHO (dextrose). Previous investigations have shown that maximal rate of exogenous carbohydrate use is about 1.0-1.1 g·min−1 when a single-carbohydrate source (e.g., dextrose) is ingested (21-23,31), but when multiple carbohydrate types (e.g., dextrose and fructose) are ingested, this rate can be increased significantly (17,19-21). Furthermore, Currell and Jeukendrup (7) demonstrated an 8% improvement in time to complete a subsequent time trial when a mixture of glucose and fructose was ingested during a previous bout of prolonged exercise compared to glucose only. The use of different stereospecific intestinal carbohydrate transporters for the different carbohydrates used is most likely the reason for the improved exogenous carbohydrate oxidation and thus, improved performance.
Therefore, the purpose of the present study was (a) to assess the efficacy of a supplement containing a mixture of carbohydrates (dextrose, maltodextrin, and fructose), yet a lower total carbohydrate content, and a moderate amount of protein (MCP, 3.0% CHO, 1.2% PRO), in extending TTE, and (b) to determine if muscle damage would be reduced by the protein-containing supplement. We hypothesized that TTE would be extended and muscle damage reduced with the MCP supplement compared to CHO, despite the MCP supplement containing 50% less carbohydrate and 30% fewer calories.
Experimental Approach to the Problem
After first determining each subject's V̇o2max, each subject completed a familiarization trial in which only water was provided, followed by 2 double-blinded, randomly ordered experimental trials in which a 6% carbohydrate beverage (CHO) or a 3% carbohydrate/1.2% protein (MCP) beverage was provided during exercise. The CHO beverage consisted of dextrose, and the MCP beverage contained dextrose, maltodextrin, and fructose (1% each), and a whey protein isolate. At the beginning of exercise and every 20 minutes thereafter, 275 mL of the selected beverage (CHO or MCP) was consumed. The subjects performed each trial in a room of ∼21°C at the same time of the day and the same day of the week over a 3-week period. Human Performance Laboratories, LLC (Austin, TX, USA) provided the beverages in powder form and were mixed in the laboratory to the concentrations specified above. The energy and macronutrient content of the beverages are shown in Table 1. The beverages were similar in color, taste, and texture to allow a double-blinded and randomly ordered study design. A laboratory technician who was not involved in the data collection prepared the beverages for each trial.
The cycling protocol for all rides is shown in Figure 1. Subjects rode the cycle ergometer at intensities alternating between 45 and 70% V̇o2max for 3 hours, and then the workload was increased to ∼74-85% V̇o2max until exhaustion. The outcome measures of interest were TTE, substrate use, and responses of insulin, glucose, lactate, and myoglobin to the different treatments.
Fifteen trained endurance athletes (cyclists and triathletes) between the ages of 20 and 40 years were admitted to the study (8 men, 7 women). Subject characteristics are listed in Table 2. All subjects were accustomed to cycling between 3 and 6 hours in a single ride. Mean years of cycling training were 6.5 ± 1.2 years. Written informed consent was obtained from each subject, and the study was approved by The University of Texas at Austin Institutional Review Board. Each subject served as his or her own control and performed the same protocol as shown in Figure 1 for each treatment.
Before beginning the 2 experimental trials, subjects reported to the laboratory for determination of their V̇o2max. The V̇o2max tests and all experimental trials were performed on the same ergometer (Velotron Dynafit Pro, Racermate, Seattle, WA, USA). The protocol for establishing V̇o2max consisted of a 4-minute warm-up, then 2-minute stages beginning at 200 W for men or 130 W for women. The workload was increased by 50 W (men) or 35 W (women) every 2 minutes until 350 and 200 W, respectively. After this point, the workload was increased by 25 W (men) or 10 W (women) every minute until the subject could not continue to pedal despite constant verbal encouragement. The criteria used to establish V̇o2max was a plateau in V̇o2 with increasing exercise intensity and respiratory exchange ratio (RER) > 1.10. During the test, subjects breathed through a Hans Rudolph valve, with expired gases directed to a mixing chamber for analysis of oxygen (O2) and carbon dioxide (CO2) (ParvoMedics TrueOne 2400, ParvoMedics, Sandy, UT, USA). Outputs from this system were directed to a laboratory computer for calculation of ventilation, O2 consumption (V̇o2), CO2 production (V̇co2), and RER every 15 seconds.
Maximum power output in watts was calculated from the V̇o2max test data using the formula, adapted from Astrand and Rodahl (1):
The workloads were then set as percentages of the Wmax as follows:
Each experimental trial was separated by a minimum of 7 days and did not exceed 14 days.
Ventilatory Threshold (Post Priori)
Post priori, we sought to determine if the intensity at which subjects cycled to exhaustion relative to their individual ventilatory thresholds (VTs) contributed to the increases in TTE. Therefore, using the minute ventilation (VE), V̇co2, and V̇o2 data from the V̇o2max test, VT was calculated using a computer-generated plot (ParvoMedics TrueOne 2400 software). Ventilatory threshold was defined as the point at which the V̇E (minute ventilation) increased in a nonlinear fashion compared to increases in V̇o2 and was substantiated by an increase in the V̇E/V̇co2 to V̇E/V̇o2 ratio. Determination of VT was performed blinded. V̇o2 was then calculated for the first 5 minutes of the performance part of the cycling protocol for each experimental trial and used to determine the percent of VT at which each subject was cycling. We then grouped the subjects as cycling at or below VT, or above VT post priori.
Three to five days after the V̇o2max test, the subjects again reported to the laboratory to perform a familiarization ride, which also allowed verification and subsequent adjustment of the calculated workloads for the experimental trials. The familiarization ride followed exactly the same protocol as the experimental rides, except that no blood samples were collected, and only water was provided every 20 minutes. The protocol is shown in Figure 1. The first 30 minutes of the protocol was at low intensity (45% V̇o2max). For the next 1.5 hours, the intensity alternated every 8 minutes between 45 and 70% V̇o2max. From hours 2 to 3, the intensity continued to alternate between 45 and 70% V̇o2max but did so every 3 minutes. After the 3-hour time point, the intensity increased to between 74 and 85% of V̇o2max (exhaustion protocol), and this marked the start of the TTE determination. Subjects were encouraged to ride as long as possible while maintaining a pedaling cadence of 80-90 revolutions per minute (rpm). When they could no longer maintain a pedaling cadence of 60 rpm despite constant verbal encouragement, they were asked to stop, and TTE was recorded as minutes:seconds beyond the 3-hour point. Constant verbal encouragement was given to the subjects during each trial, and the same investigators were present during all trials for each subject so that encouragement was consistent across all trials. In addition, subjects were not aware of how long they rode each time, because all timing devices were removed from their line of sight or covered.
For all trials, the subjects arrived at the laboratory after an overnight, 12-hour fast, during which they were allowed to consume only water. Upon arrival, body weight was obtained and a heart rate monitor (Polar Beat, Polar Electro, Oy, Finland) was secured in place around the subject's chest. For the 2 experimental trials, a catheter fitted with a 3-way stopcock and extended with a catheter extension was inserted into an antecubital vein and taped in place. A resting blood sample was taken as described below, and then the subject was given the first dose of supplement to drink. After consuming the 275 mL of beverage, the subject mounted the ergometer, and the cycling protocol began. Supplements (275 mL) were provided every 20 minutes for the duration of the ride. If the subjects were able to ride longer than 40 minutes during the exhaustion portion of the protocol (i.e., 40 minutes beyond the 3-hour ride), and felt too full to continue to drink the entire amount of supplement provided each time, then they were asked to consume as much as they felt comfortable ingesting. During the exercise trials, the laboratory temperature was maintained at ∼21°C, and a fan was directed toward the subject to reduce thermal stress.
Diet and Exercise
The subjects were instructed to maintain a training and dietary log for the 2 and 3 days, respectively, before the familiarization trial and to keep training and diet consistent with that record for the 2 and 3 days before the remaining experimental trials. The subjects provided a copy of their training and dietary logs on the day of the trials. An investigator reviewed and verified the entries in the logs with the subjects at each session to verify that compliance with the previous logs was attained. The data from the logs were entered into Nutribase Clinical Nutrition Manager 7.17 (CyberSoft, Inc., Phoenix, AZ, USA) for nutritional analysis and compliance verification. All subjects complied with the diet and exercise requirements. Diets were not standardized across all subjects, because each subject served as his or her own control.
Before receiving the first beverage dose and mounting the ergometer, a 5-ml sample of blood was collected and the catheter was flushed with saline. Five milliliters was drawn at 3 additional time points: at 118 minutes of exercise, at 177 minutes of exercise, and immediately after exercise ceased because of exhaustion. Saline flushes occurred every 10-15 minutes during the entire protocol to keep the catheter patent.
Ventilation, V̇o2, Respiratory Exchange Ratio, Heart Rate, and Ratings of Perceived Exertion
Ventilation, V̇o2, CO2 production, and RER were recorded using the same ParvoMedics TrueOne 2400 system that was used during the V̇o2max test and familiarization trial. The system was calibrated immediately before each trial using medical-grade gases of known concentrations and a 3.0-L calibration syringe. Collections were made at 4 time points: 10-15 minutes (low intensity), 46-51 minutes (high intensity), 130-136 minutes (low and high intensity, 3 minutes each) and for the first 5 minutes of the exhaustion portion. With the exception of the 130- to 136-minute collection, respiratory gases were collected for 5 minutes using 15-second sampling, and only the last 1.5 minutes of each collection were used to determine steady state V̇o2 and RER. For the 130- to 136-minute collection (3 minutes of low intensity and 3 minutes of high intensity), the last minute of each interval was used. Heart rate (HR) was recorded at the beginning of exercise and at every 10-15 minutes of exercise. Subjective ratings of perceived exertion (RPEs) on a Borg scale (ranging from 6 to 20) were obtained during exercise at the same time points as HR.
Determination of substrate (carbohydrate and fat) oxidation rates were made from V̇o2, V̇co2, and RER values using the 5 collection times as described above during the experimental rides according to the method of Frayn (11).
Biochemical Analyses of Plasma Metabolites
Each 5-mL blood sample taken during the protocol was anticoagulated with 0.3 mL of ethylenediaminetetraacetic acid (EDTA) (24 mg·mL−1, pH 7.4), and 0.5 mL of the anticoagulated blood was transferred to another tube containing 1 mL of 10% perchloric acid (PCA). All tubes were centrifuged at 4°C for 10 minutes at 3,000 rpm with a HS-4 rotor in a Sorvall RC6 centrifuge (Kendro Laboratory Products, Newtown, CT, USA). After centrifugation, plasma and PCA extracts were separated into aliquots for each assay and immediately frozen and stored at −80°C for later analysis. Plasma insulin was measured by radioimmunoassy (12) using the ImmuChem™ Coated Tube 125I RIA Kit (MP Biomedicals, LLC, Orangeburg, NY). All samples were run in duplicate, with a coefficient of variation (CV) of 6.0%. Plasma myoglobin concentrations were determined by solid phase enzyme-linked immunosorbent assay (BioCheck, Inc., Foster City, CA, USA), and were run in duplicate with a CV of 5.4%. Blood lactate was determined from the PCA extract by enzymatic-spectophotometric analysis based on the oxidation of lactate to pyruvate by nicotinamide adenine dicnucleotide according to the method of Hohorst (13). Samples were run in duplicate and had a CV of 1.5%. Plasma glucose was measured using a spectophotometric Trender reaction (no. 315, Sigma Chemical, St. Louis, MO, USA). The Trender reagent contained the enzyme horseradish peroxidase (HPOD), 4-aminoantipyrine (4-AAP) and p-hydroxybenzene sulfonate (p-HBS). Glucose was oxidized to d-gluconate by glucose oxidase with production of an equal amount of hydrogen peroxide. Coupled by hydrogen peroxide, 4-AAP and p-HBS were oxidized by HPOD and formed a quinoneimine dye, intensely colored in red. The absorbance of the reaction solution was measured using a spectrophotometer (Beckman DU 640; Beckman Coulter, Inc., Fullerton, CA, USA) at a wavelength of 500 nm. The intensity of the color in the reaction solution was proportional to the concentration of glucose in the plasma sample. Samples for this assay were also run in duplicate, with a CV of 3.7%.
The data were analyzed using a general linear model for repeated measures. Time to fatigue was analyzed using a 1-way analysis of variance (ANOVA). All the other variables that included multiple measures per trial were analyzed using a 2-way ANOVA (treatment × time). Post hoc analysis was performed when significance was found using Fisher's least square difference. The level of significance for all analyses was set at p ≤ 0.05. All data are expressed as mean ± SEM. SPSS version 16.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.
Time to Exhaustion
Time to exhaustion for the combined group, and by VT grouping, is shown in Figure 2. For the combined group (n = 15), TTE was not significantly different between treatments (MCP, 31.06 ± 5.76 vs. CHO, 26.03 ± 4.27, p = 0.064). However, for subjects cycling at or below VT (n = 8), TTE in MCP was significantly greater than CHO (45.64 ± 7.38 vs. 35.47 ± 5.94 minutes, p = 0.006). There were no significant differences in TTE for the above VT group (n = 7); CHO, 15.25 ± 2.83 vs. MCP, 14.39 ± 2.50 minutes, p = 0.8).
Blood and Plasma Analyses
Blood samples were analyzed for plasma insulin, plasma glucose, blood lactate and plasma myoglobin. No significant overall treatment differences in plasma insulin levels were found among treatments (Figure 3). However, a significant treatment by time difference was observed at 177 minutes, with plasma insulin higher in CHO than in MCP in the combined group (p = 0.023), and in the at or below VT group (p = 0.032). No significant differences were found when exercising above VT.
Plasma glucose was significantly lower in MCP than CHO in the combined group (Figure 4). Significant treatment by time effects were also observed at minutes 118 and 177 (p = 0.003 and 0.005, respectively). No treatment by time differences were found when grouped by VT.
Blood lactate levels rose significantly during the TTE portion of the protocol (Figure 5), but there were no differences between the treatments whether grouped or not grouped by VT.
Plasma myoglobin levels rose during exercise in both treatments. Although myoglobin appeared to be lower at End in MCP, no significant differences were found between treatments (p = 0.189, Figure 6). A similar nonsignificant trend was found in the group exercising above VT.
Ratings of Perceived Exertion and Heart Rate
Although RPE and HR values were recorded at the beginning of exercise and at every 10-15 minutes of exercise, 3 time points during low intensity intervals (90, 130, and 161 minutes), 2 time points during high intensity intervals (115 and 159 minutes), and 1 time point during the TTE protocol (184 minutes) were chosen for analysis. Values are shown in Table 3. There were no significant differences for RPE or HR between treatments regardless of VT grouping.
There were no significant treatment differences in either carbohydrate oxidation rates or fat oxidation rates (g·min−1) between MCP and CHO whether grouped or not grouped by VT (Table 4).
The primary objective of the present study was to compare the effects of a beverage containing a lower total amount, yet multiple types of carbohydrates, and a moderate amount of protein (3% CHO, 1.2% PRO) with a traditional 6% carbohydrate beverage on TTE during prolonged endurance exercise. The most important finding of this investigation was that TTE was significantly greater in the treatment that contained fewer calories, lower carbohydrates, and a moderate amount of protein (MCP), compared to the higher carbohydrate-containing beverage (CHO) when exercising at VT or ∼2% below VT (MCP, 45.64 ± 7.38 vs. CHO, 35.47 ± 5.94 minutes, p = 0.006). This represents a 28.7% improvement in TTE in the MCP treatment. In the combined group of 15 subjects, TTE in MCP was greater than in CHO by 19.3% (31.06 ± 5.76 vs. 26.03 ± 4.27, p = 0.064), although it did not reach statistical significance. When exercising above VT by 5-7%, there was no difference in TTE between treatments (15.25 ± 2.83 vs. MCP, 14.39 ± 2.50 minutes, p = 0.8).
Our finding of differences when grouped according to VT (e.g., cycling at or below VT or cycling above VT during the exhaustion protocol) is novel. Many investigations report TTE when cycling at a work rate that elicits a certain percentage of V̇o2max, often 70-85% V̇o2max (26-28,30), without identifying how this percentage relates to the subjects' VTs or lactate thresholds (LTs). Recently, other investigators have pointed out that the relative intensity at which individuals are exercising could be a factor in determining the efficacy of the CHO and CHO-PRO supplements (4). Indeed, the ability to exercise for long periods near an individual's LT becomes a critical component of performance in long events such as marathons, longer cycling races, and long-distance triathlons. In fact, it has been shown that runners self-select a running pace just above the point of blood lactate appearance and are able to maintain that intensity for the duration of a marathon (10). In the present study, we used VT post priori as a surrogate for LT, given that VT and LT have been shown to occur near the same V̇o2 (8,16). The significant differences found in TTE when cycling at or below VT suggest that MCP is more effective in extending endurance and delaying fatigue than CHO around the exercise intensity at which prolonged endurance performance is often crucial. Although we did not observe differences in TTE when exercising above VT, it is possible that other factors that contribute to exhaustion when exercising at higher intensities, such as a significant drop in muscle pH or depletion of high-energy phosphates, simply may not be affected by the supplement.
Many investigations have demonstrated improved TTE with the addition of protein to a carbohydrate supplement ingested during exercise (15,27,28), although the supplements are usually isocaloric or isocarbohydrate compared to a 6-8% CHO supplement (15,27). However, Martinez-Lagunas et al. (24) recently compared the effects of a 4.5% CHO plus 1.15% PRO, and a 3% CHO plus 0.75% PRO beverage to a traditional 6% CHO beverage. The investigators found that although TTE was not significantly different between treatments, both of the lower CHO plus PRO treatments still maintained TTE performance. Although their investigation used a similar exercise protocol and low CHO-PRO/low calorie treatment as were used in the present study, there are methodological differences that may have contributed to their nonsignificant differences in TTE. First, the Martinez-Lagunas et al. (24) investigation did not take into consideration the relative intensity at which the subjects were exercising. Second, Martinez-Lagunas et al. (24) used a single carbohydrate (dextrose) rather than a mixture of different carbohydrates as was employed in the present study.
The use of 3 different carbohydrates in the MCP beverage used in the present investigation may have enabled the optimization of the various intestinal carbohydrate transporters such that the rate of absorption was increased beyond that of the single-CHO treatment, leading to increased exogenous CHO oxidation and decreased endogenous CHO oxidation. Several previous investigations have demonstrated that, compared to a single-CHO supplement, supplements containing mixtures of carbohydrates can increase exogenous CHO oxidation (17-20,32), decrease endogenous CHO oxidation (17,19), and improve cycling exercise performance (7). Recently, Currell and Jeukendrup (7) compared the effects of a CHO mixture to a single-CHO supplement on cycling performance. Subjects cycled for 120 minutes at 55% Wmax while ingesting a glucose-only supplement, an isocaloric glucose plus fructose supplement, or water, followed by a time trial in which the subjects had to complete a set amount of work as quickly as possible. Ingestion of the glucose plus fructose supplement resulted in an 8% faster time trial time compared to glucose only, and 19% faster than water (7). Total CHO oxidation was not different between the CHO treatments, and the investigators concluded that ingestion of the CHO mixture likely led to a sparing of endogenous CHO stores, because glucose plus fructose has been shown to have a greater exogenous CHO oxidation than glucose only (18,19). Taken together, these findings suggest that the sparing of endogenous CHO may be a key mechanism that resulted in the TTE improvements reported here.
Others, however, have failed to show that CHO-PRO supplementation during exercise spares endogenous fuel supplies by decreasing muscle glycogen use or depletion of Krebs cycle intermediates (4). Cermak et al. (4) found no difference in muscle glycogen use or in the levels of Krebs cycle intermediates when trained men ingested 6% CHO or 6 + 2% PRO during 90 minutes of cycling at ∼69% V̇o2peak. It should be noted, however, that the investigators used a single carbohydrate in both treatments rather than a mixture of carbohydrates as was used in the present study. It is possible that using a mixture of carbohydrates in a CHO-PRO supplement could demonstrate a positive effect in the levels of muscle glycogen use and Krebs cycle intermediates compared to a supplement containing a single carbohydrate with added protein, although we did not measure these parameters in the present study.
Other investigators have proposed that reducing muscle damage that occurs during intense endurance exercise is a possible mechanism for improved performance with CHO-PRO supplementation. Saunders et al. (27,28) have demonstrated significant improvements in TTE concomitant with significant reductions in markers of muscle damage when comparing CHO-PRO supplements to CHO only supplements (27,28). Others have used a resistance exercise model to demonstrate a significant reduction in myoglobin levels 6 hours after a strenuous resistance exercise bout when subjects ingested a CHO-PRO beverage compared to a placebo supplement (2). However, other recent investigations have demonstrated significant reductions in muscle damage without differences in TTE (26,30).
In the present investigation, however, we did not demonstrate a statistically significant reduction in myoglobin. Myoglobin was selected as our muscle damage marker of choice because it is a small molecule that leaks from the skeletal muscle cell early on during exercise when damage occurs, peaking in about 1 hour postexercise (9,29). In the combined group (Figure 6), myoglobin levels continued to rise over the course of the trial with the CHO treatment, whereas with MCP, myoglobin increased to a lesser but nonsignificant extent (p = 0.189). It is possible that had we evaluated myoglobin again within hours postexercise rather than immediately at the end of exercise (e.g., at exhaustion), a greater difference in myoglobin levels may have been detected. A trend was observed for an increasing difference in myoglobin levels between treatments, yet without a measurement an hour postexercise, we do not know if that trend would have continued, resulting in a significant difference. Therefore, although we cannot associate the improvement in TTE observed in the present study with muscle damage reduction, the possibility remains that reduced muscle damage could potentially help to maintain or improve performance.
In summary, the present study demonstrated that a supplement containing a mixture of carbohydrates plus a moderate amount of protein could significantly improve aerobic endurance when cycling at or below the VT, despite containing 50% less total carbohydrate and 30% fewer calories relative to a higher carbohydrate beverage.
Proper nutritional supplementation during endurance exercise is essential for delaying fatigue and maintaining optimal performance. Most of the commercially available products contain high total calories and large amounts of carbohydrate. Many athletes and recreational exercisers want to maintain or improve their body composition and are therefore concerned about limiting caloric intake. The present investigation demonstrates that consuming a beverage containing a mixture of different carbohydrates, a moderate amount of protein and fewer calories than a traditional, higher single-carbohydrate supplement during endurance exercise can extend exercise TTE, especially when exercising at or below the VT. This is an important finding that is highly relevant to endurance athletes and exercisers, especially those who wish to consume less total carbohydrate and fewer calories and improve endurance capacity.
The authors wish to thank the subjects in this study for their time, energy, and dedication. Funding for this study was provided by HPL Laboratories LLC, Austin, TX, USA. The results of the present study do not constitute endorsement of the product by the authors or the National Strength and Conditioning Association.
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