Previous research has demonstrated that consuming a carbohydrate (CHO) supplement during prolonged endurance exercise improves performance compared to water or placebo (4,5,10,18,21,37,38). The addition of protein (PRO) to a CHO supplement, however, has demonstrated enhanced performance beyond that of CHO alone (19,29,30), but these findings are not universal (23,33).
Investigations from our laboratory recently found PRO added to either a low CHO (3% CHO + 0.75% PRO) or moderate CHO supplement (4.5% CHO + 1.2% PRO) maintained endurance performance efficacy relative to a traditional 6% CHO supplement (25). In an effort to improve the effectiveness of our CHO plus PRO supplement, we altered the CHO source from a single source of dextrose to a mixture of glucose, fructose, and maltodextrin. Multiple sources of CHO appear to increase CHO oxidation rates above that achieved by a single CHO source. The maximal rate of exogenous CHO use during prolonged exercise while providing a single CHO source is about 1.0-1.1 g·min−1 (22,35). The oxidation rate, however, can increase to 1.7 g·min−1 when a combination of CHO is ingested during exercise (20). Furthermore, the combination of multiple CHO sources has been shown to further enhance endurance performance above that of glucose alone (6,32).
We previously reported that a 3% mixed CHO plus 1.2% PRO supplement did not improve time to exhaustion (TTE) in comparison to a traditional 6% CHO treatment. However, on closer inspection, it was noted that improvement in performance did occur in subjects exercising at or below their ventilatory threshold (VT) (12). Based on these findings, we sought to determine if this improved performance could be further demonstrated when utilizing a larger subject sample exercising at an intensity approximating their VT.
Therefore, the purpose of this study was to investigate if the addition of a moderate PRO (1.2%) concentration to a low CHO (3%) mixture (glucose, maltodextrin, and fructose) (CHO + PRO) would improve TTE in comparison to a traditional 6% CHO supplement, in female athletes exercising at or slightly below their VT. It was hypothesized that performance would be enhanced when consuming CHO + PRO in comparison to a traditional 6% CHO supplement, despite a 50% lower CHO concentration and 33% lower caloric intake with the CHO + PRO treatment.
Experimental Approach to the Problem
This study followed a randomized, double-blinded, repeated-measures design. After initially completing a maximal oxygen consumption (V̇O2max) test and familiarization trial, subjects performed 2 experimental trials separated by 1 week. The experimental protocol was composed of varying intervals between 45 and 70% V̇O2max, followed by a ride to exhaustion at an intensity approximating the individual's VT. Supplements (275 mL) were consumed immediately before commencing the trial, and every 20 minutes thereafter.
The CHO + PRO contained a mixture of dextrose (glucose), maltodextrin and fructose, and a whey PRO isolate. The CHO treatment was composed of dextrose only. CHO + PRO contained 50% the CHO content in comparison to CHO and 33% lower caloric content. Both treatments contained equal amounts of electrolytes (Table 1). Supplements were supplied by the Human Performance Laboratory (Austin, TX, USA) and were prepared by a laboratory technician not directly involved in the study. All supplements were similar in taste, color, and texture.
Fourteen female cyclists and triathletes were recruited via e-mail announcement from local triathlon and cycling teams in Austin, TX, USA. A detailed explanation of the experimental procedures and the potential risks of the study were given both verbally and in writing to all subjects before initial testing. Subjects were given the opportunity to ask questions before signing the informed consent, according to the protocol described in the University of Texas at Austin's ‘Institutional Review Board Procedures Manual for Faculty, Staff and Student Researchers with Human Participants.’ The University of Texas at Austin Institutional Review Board approved the study before it commenced. Subject characteristics are found in Table 2.
Subjects initially reported to the laboratory for determination of V̇O2max and VT. All trials were conducted on the same cycle ergometer (Veletron Dynafit Pro, Racermate, Seattle, WA, USA). Before testing, body weight was recorded and subjects were outfitted with a Polar heart rRate (HR) monitor (Polar Beat, Polar Electro, Oy, Finland).
Maximal oxygen consumption was determined via a ramped protocol, consisting of a 4-minute warm-up stage (range 75-130 W), followed by 4 stages of 2-minute duration. Each 2-minute stage increased in intensity by 35-W increments. Thereafter, workloads increased by 10 W each minute until the subject could no longer continue. Subjects breathed through a Hans Rudolf valve, with expired gases directed to a mixing chamber for analysis of oxygen and carbon dioxide. Inspired air volumes were measured using a dry gas meter (ParvoMedics TrueOne2400, ParvoMedics, Sandy, UT, USA). A laboratory computer collected gas meter outputs, and used values for calculation of oxygen uptake (V̇O2), carbon dioxide production (VCO2), and Respiratory exchange ratio (RER) every 15 seconds. The criteria for establishing V̇O2max were a plateau in V̇O2 with increasing exercise intensity in addition to an RER > 1.10. The 2 highest 30-second values were averaged to determine V̇O2max (ml O2·kg−1·min−1). The VT was determined from a computer generated plot of values obtained during the V̇O2max test (ParvoMedics TrueOne2400 software). ventilatory threshold was defined as the point of a nonlinear increase in minute ventilation (V̇E) in comparison to increases in V̇O2. This was confirmed by an increase in the V̇E/V̇CO2 to V̇E/V̇O2 ratio.
All trials were conducted in the Exercise Physiology Metabolism Laboratory at The University of Texas at Austin. Within the 7 days after preliminary testing, subjects reported to the laboratory for a familiarization trial. This trial simulated the experimental protocol, exclusive of blood draws and treatment beverages. Water (275 mL) was substituted for the experimental beverages.
The cycling protocol consisted of varying intervals between 45 and 70% V̇O2max, followed by a ride to exhaustion at an exercise intensity approximating the individual's VT. The first 30 minutes of cycling was conducted at 45% V̇O2max, followed by 6 intervals of 8-minute duration. Interval duration was then reduced to 3 minutes. At 3 hours into the cycling protocol, subjects began the performance ride at an intensity relative to their VT, and this intensity was held until exhaustion. (Refer to Figure 1 for the cycling protocol.) Exhaustion was determined as the point at which subjects could no longer maintain a pedaling cadence of 60 rpm, despite constant verbal encouragement.
On the morning of the experimental trials, subjects arrived at the laboratory between 7 and 8 am, after a 12-hour fast during which they were permitted to consume water only. Diet and activity logs were collected and verified, and body weight was obtained. Subjects were fitted with a Polar HR monitor and a Teflon catheter, fitted with a 3-way stopcock and a catheter extension, was inserted into an antecubital vein. Subjects were instructed to sit quietly for 2 minutes, after which a baseline HR was recorded and a 5-mL baseline blood draw was taken. After baseline blood sampling, participants consumed the first supplement (275 mL) before mounting the ergometer. Supplements were provided every 20 minutes during the exercise protocol. Upon nearing exhaustion, however, subjects were asked to consume only as much as they felt comfortable.
All timing devices were removed from the subject's sight, blinding participants to the length of ride completed. Personal music devices were permitted; however, devices were required to be on a random song shuffle setting to eliminate any indication of time.
Cardiorespiratory Measures, Ratings of Perceived Exertion
Respiratory gas samples and ventilation were collected 5 different times throughout the protocol. Collections occurred at 10 minutes (low intensity), 46 minutes (high), 130 minutes and 184 minutes (start of exhaustion ride). Collection periods were 5 minutes in length, excluding the collection at time point 130-136 minutes, which consisted of 2, 3-minute recordings at low and high intensities, respectively. To ensure a steady-state V̇O2 and RER, only the last 2 minutes of each collection was recorded. Carbohydrate and fat oxidation rates (g·min−1) were calculated from V̇CO2, V̇O2, and RER according to Frayn (13). It was assumed that PRO oxidation during exercise was negligible. The HR and rating of perceived exertion (RPE) were recorded 12 times throughout the exercise protocol. The RPE was recorded using the Borg Scale (2).
Blood samples (5 mL) were collected pre-exercise (PRE), 118 minutes (T −118) and 177 minutes (T − 177) into the exercise protocol, and at the point of exhaustion (END). The sample (0.3 mL) was transferred into a separate tube containing 1 mL 10% perchloric acid (PCA). The remaining sample was divided into 2 tubes and mixed with 0.3 mL of EDTA (24 mg·mL−1, pH 7.4) to prevent coagulation. Tubes were centrifuged for 10 minutes at 3,000 rpm in a HS-4 rotor in a Sorvall RC6 centrifuge (Kendro Laboratory Products, Newtown, CT, USA). Plasma and PCA extracts were transferred, and all tubes were stored at −80°C until analysis.
Prior Diet and Exercise
Subjects were required to record activity levels for 3 days and diet for the 2 days before each trial. Diet and exercise were recorded in supplied logs, and subjects were required to replicate diet and exercise before each trial. Subjects were asked to keep diet and activity levels as close to their regular routine as possible and asked to refrain from strenuous exercise in the 24 hours before the trial. Logs were reviewed before each trial to ensure compliance. All subjects abided by the requirements.
Biochemical Analyses of Plasma Metabolites
At each blood collection, 1 drop of blood was used to measure blood glucose concentration (One Touch Basic glucose analyzer, LifeScan Inc., Milipitas, CA, USA). This sample was used to ensure subjects were fasted upon arrival and additionally, as an indicator of blood glucose levels as the trial progressed. Before each trial, the analyzer was calibrated using standards provided by LifeScan Inc.
For data analysis, plasma samples were measured for glucose in duplicate using a modified Trinder procedure at 37°C (31). Samples were read at 500 nm using a Beckman DU640 Spectrophotometer (Coulter, Fullerton, CA, USA) and had a coefficient of variation (CV) of 3.7%.
Blood lactate concentrations were measured from the PCA extracts using enzymatic analysis according to Hohorst (17). The assay was run in duplicate and had a CV of 1.2%. Samples were read at 340 nm using a Beckman DU640 Spectrophotometer (Coulter).
Plasma insulin was analyzed via radioimmunoassay (RIA) based on the principles of Goetz and Greenberg (15) (MP Biomedicals 125I RIA, Solon, OH, USA) and had a CV of 6.0%. Duplicate tubes were prepared and counted in a Wallac 1470 Wizard Gamma Counter (PerkinElmer Life and Analytical Sciences, Boston, MA, USA), which had been calibrated for counting 125I.
Myoglobin was measured in duplicate by a solid-phase enzyme-linked immunosorbent assay (myoglobin enzyme immunoassay test Kit, BioCheck, Inc, Foster City, CA, USA), with a CV of 5.4%. Wells were read at 450 nm with a microtiter well reader (Bio-Tek ELx800, Biotek Instruments Inc, Winooski, VT, USA).
Data were analyzed using SPSS for Windows, version 16.0 (SPSS Inc., Chicago, IL, USA). The TTE was analyzed using a paired t-test. Average HR and substrate (CHO and fat) use across the variable intensity protocol was compared between trials using a paired t-test. All other variables were measured using a 2-way (treatment × time) repeated-measures analysis of variance. Where significance was found, post hoc comparisons were conducted using a least significant difference adjustment. Significance was determined at p ≤ 0.05. Data were expressed as mean ± SE.
Time to exhaustion was significantly greater with CHO + PRO, with a 15.2% increase in performance in comparison to CHO (CHO + PRO: 49.94 ± 7.01 vs. CHO: 42.36 ± 6.21 minutes, p < 0.05) (Figure 2). Subjects performed the exhaustion ride at an average of 75.06% V̇O2max, ∼1.5% lower than the calculated average group VT (76.57 ± 1.24%V̇O2max). Intensities for individual subjects ranged from 7.25% below VT to 5.1% above VT.
Blood and Plasma Analyses
There were no significant differences in the pre-exercise plasma glucose levels. Plasma glucose levels were increased significantly from PRE to END only in the CHO treatment. Additionally, mean blood glucose for CHO (4.47 ± 0.12 mmol·L−1) was significantly greater than for CHO + PRO (4.07 ± 0.12 mmol·L−1) with treatment by time differences occurring at minutes 118 and 177, and at the point of exhaustion (p < 0.05) (Figure 3).
Plasma insulin levels decreased as exercise progressed during both exercise trials, and there were no significant differences between treatments (Figure 4). Average plasma insulin was 73.56 ± 7.37 pmol·L−1 during the CHO trial and 70.00 ± 7.78 pmol·L−1 during the CHO + PRO trial.
Average blood lactate concentration was 1.21 ± 0.139 mmol·L−1 for the CHO treatment and 1.22 ± 0.147 mmol·L−1 for the CHO + PRO treatment. No significant differences were found between treatments or treatment by time (Figure 5). In both treatments, blood lactate concentrations significantly increased from 177 minutes to exhaustion (p < 0.05).
The average plasma myoglobin concentration was 26.28 ± 7.28 ng·mL−1 during the CHO trial and 19.64 ± 1.79 ng·mL−1 during the CHO + PRO trial. However, statistical analysis revealed no significant overall difference in treatment or treatment by time interaction for myoglobin (Figure 6).
Respiratory Exchange Ratio and Substrate Use
The average RER across the first 3 hours of the CHO treatment was 0.924 ± 0.011 and 0.939 ± 0.012 for CHO + PRO (Table 3). V̇O2 over the same period was slightly, but significantly higher during the CHO + PRO trial (1.779 L·min−1) as compared to CHO (1.755 ± 0.09 L·min−1, p < 0.05). Carbohydrate and fat use were calculated from V̇O2, V̇CO2, and RER data. Collections occurred only during the 3-hour variable intensity ride; thus, results are not indicative of substrate use rates during the performance ride to exhaustion. Average CHO oxidation was 1.76 ± 0.12 g·min−1 for the CHO trial and 1.75 ± 0.12 g·min−1 for the CHO + PRO trial. Average fat oxidation for the CHO and CHO + PRO trials were 0.24 ± 0.04 and 0.22 ± 0.04 g·min−1, respectively. There were no significant treatment differences in either CHO or fat oxidation rates (g·min−1) between CHO + PRO and CHO treatments (Table 3).
Heart Rate and Ratings of Perceived Exertion
During exercise, average HR was significantly lower during the CHO + PRO (130.17 ± 3.13 b·min−1) trial in comparison to CHO (132.80 ± 2.92 b·min−1, p < 0.05). There were no significant differences between treatments for RPE (Table 4).
The primary purpose of this study was to determine if a moderate PRO low mixed CHO sports drink could increase endurance performance in comparison with a traditional 6% CHO sports drink in trained female athletes. The primary finding was that CHO + PRO enhanced TTE above that of CHO when exercising at an intensity at or slightly below VT (CHO + PRO, 49.94 ± 7.01 minutes vs. CHO, 42.36 ± 6.21 minutes, p < 0.05). This represents a 15.2% improvement in performance with CHO + PRO. Improvement in TTE occurred despite CHO + PRO containing a 50% lower CHO content and approximately 30% fewer calories. This may be an important consideration for individuals concerned about body weight and caloric intake.
This study is in agreement with previous findings that the addition of PRO to a CHO supplement enhances endurance performance in comparison with a traditional 6% CHO supplement (19,29,30). Our laboratory recently found that a low CHO plus PRO sports drink maintained efficacy in comparison to a traditional 6% CHO sports drink (26). In an effort to improve our CHO + PRO sport drink, we altered the CHO source to contain a mixture of glucose, fructose, and maltodextrin, rather than a single CHO source (glucose). We recently compared the effects of our 3% mixed CHO plus 1.2% PRO sports drink with a traditional 6% CHO sports drink during variable intensity cycling to exhaustion (12). Improvements in performance, however, were observed only in those individuals performing at or below their VT during the exhaustion portion of the cycling protocol. Recently, it was suggested that the performance effects of CHO + PRO supplementation may be related to the intensity of exercise (3). In this study, we sought to further determine whether the improved performance with CHO + PRO was related to exercise intensity.
Individuals completed the performance ride at an average of 75.06% V̇O2max, approximately 1.5% lower than the average VT (76.57 ± 1.24% V̇O2max). Individualizing the performance ride to the subject's VT is novel in comparison to prior studies investigating the performance effect of CHO + PRO supplementation. Time-to-exhaustion rides have used intensities ranging from 70 to 85% V̇O2max (19,29). Although workloads were adjusted relative to an individual's V̇O2max, these studies did not account for individual differences in VT or corresponding lactate threshold (LT). Performance during endurance sporting events, such as marathons, is evaluated at self-selected intensities that approximate LT (11). Therefore, adjusting performance trials relative to LT or VT may potentially decrease some of the variability in results across subjects.
In this study, performance was defined as the point at which individuals could no longer maintain their cycling cadence above 60 rpm during an exhaustive exercise bout. Previous criticisms of exhaustive exercise bouts are that they are not as representative of sporting events in comparison to a set distance time trial (7,33). However, supplementation benefits are not limited to endurance races such as marathons, distance road cycling, and long-distance triathlons. High levels of endurance are required in numerous situations inside and outside the sporting world. Sports such as tennis, volleyball, and baseball have the capacity to last many hours, with success reliant on lasting endurance during the later stages. Professionals such as firefighters and military personnel are routinely required to maintain high levels of physical and mental performance for prolonged periods, and prolonging TTE can be critical for the success of their mission or even survival.
The improvement in performance with CHO + PRO over CHO is potentially explained by a number of mechanisms. Exogenous CHO alone has previously demonstrated increased glucose uptake above that of placebo (1,24) and suggested to be associated with sparing of endogenous CHO (38). Insulin and muscle contraction are considered the major stimulators of glucose transport (16) and both CHO and PRO have been shown to have a stimulatory effect on insulin levels. Greater insulin response has been found with the combined ingestion of CHO with either PRO (39) or amino acids (34). However, plasma glucose uptake has been shown to be stimulated independently of insulin in the presence of the amino acids, leucine, and isoleucine in animal models (8,9,27). Additionally, CHO + PRO supplementation in humans has demonstrated further stimulation of leg glucose uptake above a CHO only treatment (25). Levenhagen et al. (25), found subjects had a 3.5-fold increase in leg glucose uptake when consuming a CHO plus PRO treatment immediately postexercise, in comparison to CHO only. Treatments were iso-CHO, thus the 3.5-fold increase in glucose uptake with CHO + PRO was likely attributed to the PRO content of the treatment.
In this study, plasma glucose levels were significantly lower during exercise as compared to CHO. However, plasma insulin levels were similar between trials; therefore, the lower plasma glucose levels cannot be attributed to an increase in insulin availability. The combination of CHO and PRO could have increased glucose clearance from the blood at a greater rate than CHO alone, resulting in lower blood glucose levels and increased exogenous CHO availability to the working muscle. However, it is also possible that the lower plasma glucose values of the CHO + PRO treatment were associated with its lower CHO concentration. The CHO + PRO treatment delivered CHO at a rate of 24.75 g CHO·h−1 (0.413 g CHO·min−1), in comparison to 49.5 g CHO·h−1 (0.83 g CHO·min−1) in the CHO treatment.
The CHO + PRO supplement in this study used a mixture of CHO sources. CHO + PRO was composed of equal amounts of dextrose, maltodextrin and fructose, as opposed to the single source of dextrose in the 6% CHO treatment. Oxidation rates up to 1.7 g·min−1 can occur when ingesting a combination of CHO at a high rate of 2.4 g·min−1 (20), in comparison a maximum rate of 1.0-1.1 g·min−1 when ingesting a single CHO source (22,35). The increased exogenous oxidation with the multiple CHO sources appears to be related to the use of different intestinal transporters. Glucose and its derivatives are absorbed into the small intestine via the sodium-dependent glucose cotransporter, whereas fructose absorption uses GLUT 5.
In comparison to a single CHO, mixed CHO supplements have previously demonstrated improved time trial performance (6,32). Currell and Jeukendrup (6) found cyclists, who initially cycled for 120 minutes at 55% V̇O2max and then competed in a time trial, had an 8% time trial improvement while ingesting a glucose plus fructose mixture, in comparison to glucose only. Similar results were seen in a recent study comparing iso-CHO glucose and glucose plus fructose during a 100-km cycling time trial (32). As in the study by Currell and Jeukendrup (6), the glucose + fructose mixture improved performance by 8% in comparison to glucose only. Previous investigations in our laboratory have additionally found improved efficacy when using a mixture of CHOs, in combination with a moderate PRO concentration (12). Therefore, it appears this is a likely mechanism contributing to the improved TTE we observed.
Heart rate during the CHO + PRO trial (130.17 ± 3.13 b·min−1) was slightly, but significantly lower, during the 3-hour variable intensity ride, as compared to CHO (132.80 ± 2.92 b·min−1, p < 0.05). However, this cannot be attributed to subjects working at a lower intensity during the CHO + PRO trial, as evidenced by a significantly higher treatment effect for V̇O2 during the same exercise period (CHO: 1.755 ± 0.09 vs. CHO+PRO: 1.779 L·min−1, p < 0.05). Potentially, this difference could be explained by a greater efficiency of the heart during the CHO + PRO trial; however, we are unable to conclude the exact mechanism behind this result. The higher V̇O2 during exercise does give evidence that the improved TTE with CHO + PRO was not related to subjects riding at a lower intensity during the stages preceding the time-to-exhaustion exercise bout.
During this study, we measured plasma myoglobin levels to indirectly assess muscle damage. Previous studies have found CHO + PRO supplementation can decrease muscle damage response to exercise (29,30). This mechanism to enhance performance was first proposed by Saunders et al. (29), when a CHO + PRO supplementation enhanced TTE by 29% compared with a traditional 6% supplement. In addition, levels of the muscle damage marker creatine phosphokinase (CPK) were found to be 83% lower 20-24 hours postexercise after CHO + PRO supplementation. In a subsequent exhaustive exercise bout 12-15 hours later, performance was 40% greater in comparison to CHO only supplementation. In a later study, Saunders et al. (30) found that subjects cycled 13% longer when consuming a CHO + PRO gel compared to CHO alone during a ride to exhaustion at 75% V̇O2peak. Plasma CPK levels were significantly lower 12-15 hours postexercise in the CHO + PRO treatment in comparison to CHO. Despite these findings, reduced muscle damage with CHO + PRO supplementation has not always corresponded with improved performance. In contrast to previous findings by Saunders et al. (29,30), we found no difference in markers of muscle damage between treatments. However, measures were only taken during and immediately postexercise, with the last collection taken at the point of exhaustion. Therefore, it is possible that the times we selected to assess muscle damage via measurement of plasma myoglobin were inadequate.
Another mechanism by which CHO + PRO may have enhanced performance is related to aerobic energy production. It has been proposed that consuming a CHO + PRO supplement during exercise maintains Krebs cycle intermediates and aerobic energy production and may enhance endurance performance (19,36). Krebs cycle intermediates increase at the onset of exercise and progressively decline as exercise continues (14,28). A decrease in CHO availability has been proposed to further decrease Krebs cycle intermediates, possibly limiting the mitochondria's ability to maintain aerobic energy capacity (28). Maintaining Krebs cycle intermediates is critical in the maintenance of aerobic energy production (28,36). The addition of PRO to CHO may further enhance CHO ability to maintain Krebs cycle intermediates during exercise (36). A study recently conducted by Cermak et al. (3) found no difference in the Krebs cycle intermediates citrate and malate while ingesting a 6% CHO plus 2% PRO or an isocarbohydrate CHO treatment. However, there was no measure of α-ketoglutarate, which is likely to be the rate limiting Krebs cycle intermediate during prolonged exercise bouts. Although not directly assessed in this study, it is possible improved endurance performance with CHO + PRO could be attributed to its ability to maintain aerobic energy capacity.
In summary, the addition of a moderate PRO concentration to a low concentration CHO mixture improved endurance performance in comparison to a traditional 6% CHO sports drink in trained female athletes. This improvement occurred despite CHO + PRO containing 50% less CHO and approximately 30% fewer calories than the traditional 6% CHO supplement. It is likely the greater performance seen with CHO + PRO was a result of the combination of PRO and the use of a mixture of CHO sources.
Consuming a beverage high in CHO concentration during endurance has demonstrated improved endurance performance. However, these beverages are often high in caloric content. In this study, CHO + PRO improved performance despite containing 50% lower CHO content and approximately 30% fewer calories. This may be an important consideration for those individuals concerned about body weight and caloric intake.
The authors wish to thank the study participants for their time and dedication to this study. Funding for this study was provided by HPL Laboratories LLC, Austin, TX, USA. The results of this study do not constitute endorsement of the product by the authors or the National Strength and Conditioning Association.
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