Quercetin supplementation increases muscle oxidative capacity and endurance in mice, but its ergogenic effect in humans has not been established. Our study investigates the effects of short-duration chronic quercetin supplementation on muscle oxidative capacity; metabolic, perceptual, and neuromuscular determinants of performance in prolonged exercise; and cycling performance in untrained men. Using a double-blind, pretest-posttest control group design, 30 recreationally active, but not endurance-trained, young men were randomly assigned to quercetin and placebo groups. A noninvasive measure of muscle oxidative capacity (phosphocreatine recovery rate using magnetic resonance spectroscopy), peak oxygen uptake (V̇o2peak), metabolic and perceptual responses to submaximal exercise, work performed on a 10-min maximal-effort cycling test following the submaximal cycling, and voluntary and electrically evoked strength loss following cycling were measured before and after 7–16 days of supplementation with 1 g/day of quercetin in a sports hydration beverage or a placebo beverage. Pretreatment-to-posttreatment changes in phosphocreatine recovery time constant, V̇o2peak, substrate utilization, and perception of effort during submaximal exercise, total work done during the 10-min maximal effort cycling trial, and voluntary and electrically evoked strength loss were not significantly different (P > 0.05) in the quercetin and placebo groups. Short duration, chronic dietary quercetin supplementation in untrained men does not improve muscle oxidative capacity; metabolic, neuromuscular and perceptual determinants of performance in prolonged exercise; or cycling performance. The null findings indicate that metabolic and physical performance consequences of quercetin supplementation observed in mice should not be generalized to humans.
- muscle oxidative capacity
- oxygen consumption
3,3′,4′,5,7-pentahydroxyflavone (quercetin) is a naturally occurring dietary polyphenolic flavonoid found primarily in skins of fruits, leafy vegetables, and berries, as well as in black tea, red wine, and various fruit juices. National dietary assessments indicate that the mean intake of quercetin in the habitual diet typically varies from < 5 mg to ∼40 mg, but daily levels as high as 200–500 mg may be attained by heavy consumers of fruits and vegetables (17). Quercetin has antioxidant, anticarcinogenic, anti-inflammatory and cardioprotective properties (17), reduces the susceptibility to viral infection (12, 42), and decreases the risk of cancer and several chronic diseases (30). Because of quercetin's healthful properties, it is marketed as a dietary supplement and added to foods and beverages.
Recently, it has been reported that quercetin feedings stimulate mitochondrial biogenesis in mice. Davis et al. (11) found that 1 wk of quercetin feedings increased mRNA of coactivators of mitochondrial biogenesis [sirtuin 1 and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α)], mitochondrial protein (cytochrome c) concentration and DNA in brain and skeletal muscle, and increased run time to exhaustion by 36–37% in sedentary mice. These findings are similar to those reported for another polyphenol, resveratrol (28). Transgenic sedentary mice with increased skeletal muscle PGC-1α exhibit greater mitochondrial content, peak rate of oxygen consumption, and exercise capacity compared with wild-type mice (8). These studies on mice indicate that polyphenols, such as quercetin and resveratrol, have effects on skeletal muscle, aerobic capacity, and endurance that mimic those of exercise training (20) and have raised the possibility that they too may counter diseases related to mitochondrial dysfunction (5, 44) and may be ergogenic in humans. However, it remains to be established whether or not the adaptations to quercetin reported for mice are observed in humans.
Findings from the few research studies on the ergogenic effects of chronic (29, 36, 37) and acute (9) quercetin supplementation in humans are equivocal. Limited data also exist on the effect of quercetin supplementation on muscle oxidative capacity (37), V̇o2peak, and substrate utilization during submaximal exercise, which would be expected to change consequent to mitochondrial biogenesis and contribute to increased endurance performance in humans with quercetin supplementation (20). The studies of chronic quercetin supplementation on muscle oxidative capacity and performance were conducted on highly trained cyclists and runners, who may have reached a ceiling for mitochondrial density due to their high level of aerobic training. Additional study of the effect of quercetin supplementation on muscle oxidative capacity, V̇o2peak, substrate utilization during exercise, and performance in untrained individuals who might experience an increase in muscle oxidative capacity is warranted.
Quercetin also might improve physical performance because of its antioxidant properties. It has been hypothesized that dietary antioxidant supplementation may improve exercise performance by minimizing damage to membranes, and contractile and structural proteins in skeletal muscle, thereby limiting the acute negative, fatiguing effects of increased reactive oxygen species generated during exercise (29). If this is true, antioxidant supplementation might reduce muscle damage, soreness, and impaired neuromuscular function that may occur during, and be evident following, exercise. Although the literature on the effect of antioxidants on exercise performance is equivocal (40), some studies suggest soreness (37) and neuromuscular function impairment (16) might be improved. Others have found no effect of quercetin on muscle soreness or neuromuscular function following exercise (36, 45). No studies have determined whether quercetin supplementation reduces loss of muscular strength that occurs with prolonged exercise (10).
Quercetin has been shown in in vitro tests to be an adenosine A1-receptor antagonist (1) and may have analgesic effects like caffeine. Thus, it could improve physical performance during prolonged exercise by reducing the perception of effort and pain, as occurs with caffeine (10, 15, 41). Quercetin also may have direct effects on muscle, augmenting force-producing capability (23). However, results from studies on cyclists and runners (29, 46) on the effect of chronic quercetin supplementation on ratings of perceived exertion (RPE) during prolonged self-paced exercise are equivocal. One study (9) found that acute supplementation with quercetin did not change RPE during exercise in the heat.
The objective of our study was to investigate the effects of short-duration, chronic quercetin supplementation on muscle oxidative capacity; metabolic, neuromuscular, and perceptual determinants of performance in prolonged exercise; and cycling performance in untrained men. It was hypothesized that short-duration dietary quercetin supplementation would increase muscle oxidative capacity, V̇o2peak, the use of fat for fuel during submaximal exercise, and cycling performance; decrease RPE during submaximal exercise; and blunt the loss of strength following prolonged exercise in untrained men.
Thirty young, healthy, recreationally active, but not endurance-trained, men participated in the study. Untrained individuals were chosen as participants to increase the probability of detection of an effect of quercetin on skeletal muscle oxidative capacity, which has been shown to increase in sedentary mice (11) but not in highly trained cyclists (37). Only men were used to reduce heterogeneity of the outcome variables, increase statistical power, and avoid confounding effects of the menstrual cycle. The protocol was approved by the University's Institutional Review Board. Participants gave written informed consent and were paid for participation.
A randomized, double-blind, pretest-posttest control group design was used. A repeated-measures, cross-over design, with greater statistical power, was not used because of length of time needed to assess all of the pretest and posttest measures, and the unknown length of time needed for washout of the effects of chronic quercetin ingestion (e.g., quercetin accumulation in tissues, potential mitochondrial adaptations). Participants were randomly assigned to an experimental (quercetin, Q) or placebo (P) group. Outcome variables were measured on both groups before and after the treatment period. The minimum duration of the treatment period (7 days) was based on the estimated minimum time needed for quercetin to accumulate in and act on tissues of interest (e.g., skeletal muscle and brain) (12). Davis et al. (11) found quite large increases in markers of mitochondrial biogenesis in skeletal muscle and brain and in endurance, all occurring with 1 wk of quercetin supplementation in mice.
During the treatment period, participants in Q ingested a sports hydration beverage prepared by The Coca-Cola Company (Atlanta, GA), containing carbohydrate (sucrose and maltodextrins), NaCl, vitamins (niacin, B6, B12), citric acid, a gel-forming additive, and quercetin (1,000 mg/day), whereas untreated participants in group P ingested the same beverage without quercetin. Investigators and participants were blinded to the treatment identity until data collection and analysis were completed. One quarter of the contents of one 946-ml nondescript bottle of the beverage identified by a code unknown to the investigators and participants was ingested four times daily, with the morning, midday, and evening meals, and prior to sleep. The purpose of the multiple feedings was to maintain elevated blood levels of total quercetin throughout the day (34). Participants were asked to report by e-mail at the end of each day to verify ingestion of the treatment.
Individual participants remained on the treatment for 9–16 days, beginning immediately after the pretests and continuing until all the posttests were administered. Pretests and posttests required three separate days for administration; thus, some participants remained on the treatment longer than others. The length of the treatment also varied among tests. MRI posttests were administered after 7 days of treatment in all participants. V̇o2peak posttests in Q and P were administered after an average of 8.2 ± 0.4 (range, 8–9) and 8.6 ± 1.9 (range, 7–14) days of treatment, and the cycling performance tests were administered after an average of 13.0 ± 1.7 (range, 10–16) and 13.0 ± 2.3 (range, 9–16) days of treatment, respectively. Participants abstained from caffeine, nonprescription drugs, vitamins, or other dietary supplements beginning 3 days prior to the pretreatment tests and continuing throughout the treatment, but maintained their normal diet and physical activity during the treatment period.
To evaluate the ergogenic effects of dietary quercetin supplementation, performance on a 10-min cycling test in which participants performed as much work as possible, following 1 h of cycling at a moderate intensity, was evaluated. In addition, maximum voluntary and electrically evoked strength loss following prolonged cycling were assessed to provide additional information on the effects of quercetin on neuromuscular function. Physiological measures during steady-state cycling prior to the performance test provided data that indicated whether changes may be due to altered substrate utilization or movement economy. RPE during steady-state cycling indicated whether performance improvements are related to effects of the supplement on adenosine receptors that might alter perception of effort. The rate of phosphocreatine (PCr) recovery following quadriceps exercise measured by nuclear magnetic resonance spectroscopy (MRS) was used to evaluate changes in muscle oxidative capacity. V̇o2peak was measured to assess any changes in whole body aerobic capacity.
Participants attended a preliminary test session at which physical characteristics and body composition were assessed and procedures used in assessment of outcome measures were practiced. Body height was measured using a wall stadiometer, body mass was determined using an electronic scale (model FW-150KA1; A&D, Tokyo, Japan), and body composition was estimated using dual-energy X-ray absorptiometry (iDXA; GE Healthcare-Lunar, Madison, WI). Participants then practiced the cycling performance test. After the cycling test, maximal voluntary and electrically stimulated strengths of right knee extensors were measured to familiarize participants with these tests.
Muscle oxidative capacity was evaluated noninvasively from the rate of creatine phosphate (PCr) recovery following brief (39 s) isometric quadriceps exercise using 31P-MRS with a 3-Tesla whole body magnet (GE Healthcare, Waukesha, WI) as described previously (2, 31–33). The rate of PCr recovery is directly related to citrate synthase activity in the gastrocnemius muscle of rats (38) and humans (31) and increases with exercise training (16, 37). To measure PCr recovery, an 1H and 31P dual-tuned radio-frequency coil (Clinical MR Solutions, Brookfield, WI) was placed on the vastus lateralis of the participant's right quadriceps. The 31P coil (a square loop 13 cm × 13 cm) overlapped the 1H coil (two loops, side by side, 20-cm diameter). A manual shimming on 1H was applied to further improve the magnetic field homogeneity and reduce spectrum distortion after an autoshimming by a prescan sequence. A free induction decay chemical shift imaging pulse sequence was applied to acquire the 31P spectrum. The scan parameters were: repetition time = 3 s, field-of-view = 18 cm, slice thickness = 10 cm, number of excitation = 1, rf pulse = hard. Following 1 min of rest, the participant performed quadriceps isometric contraction for 39 s with the leg fully extended to deplete PCr. The participant then remained as still as possible for ∼6 min of recovery. 31P-MRS data were collected before, during, and after the exercise. Three bouts of exercise were performed over the course of 30 min. The area of the PCr peaks during recovery was determined using a computer integration program, and the time constant (τ) was calculated by fitting PCr recovery with a customized Matlab (The Mathworks, Natick, MA) program to a single exponential curve: PCr = End-Delta*Exp (−Time/τ), where End, Delta, and τ are three fitting parameters of the exponential curve.
V̇o2peak. A cycling graded exercise test was conducted to measure V̇o2peak. Participants cycled on an electronically braked ergometer (Lode Excalibur Sport; Lode, Groningen, NL) with the power output starting at ∼50% of the V̇o2peak and progressively increasing by 25 W every 2 min until they could no longer continue. Oxygen uptake (V̇o2) and related gas exchange measures were obtained by open circuit spirometry using a Parvo Medics TrueOne 2400 Metabolic Measurement System (Parvo Medics, Salt Lake City, UT) and averaged over 1-min intervals. Standard gases of known composition were used to calibrate the oxygen and carbon dioxide analyzers, and a 3-liter syringe was used to calibrate the pneumotachometer prior to each test session. Heart rate (HR) and RPE were measured every 2 min and at the end of the test. HR was measured with a Polar Vantage XL HR monitor (Polar Electro, Woodbury, NY). RPE was measured by the Borg 15-point category scale following standardized instructions (7).
Experimental protocol practice.
At a second preliminary test session, procedures used in the experimental protocol were practiced. First, maximal voluntary and electrically stimulated strengths of the right knee extensors were measured to determine preexercise strength levels. Then, participants cycled on the Lode ergometer continuously for 60 min at a work rate designed to elicit 50% V̇o2peak, while V̇o2 was measured intermittently to verify the rate of oxygen uptake. Following completion of the 60-min ride and a 1-min rest period to reprogram the ergometer, participants completed a 10-min cycling performance trial in which the subject performed as much work as possible (second performance test practice). During the submaximal cycling and performance trial, participants were familiarized with use of the RPE scale. Twenty minutes after the prolonged cycling and 10-min performance test, the maximal voluntary and electrically stimulated strengths of right knee extensors of the participants were remeasured to determine the loss of muscle strength due to prolonged exercise.
Prolonged cycling and performance tests.
Participants refrained from exercise for 48 h before testing. Food records were kept for 2 days prior to and for the day of the pretest session, and this diet was replicated for the posttest session. Physical activity during the week prior to the test was assessed with the 7-day physical activity recall questionnaire (6). Participants reported to the laboratory in a normally hydrated condition 2 h after a light, mixed meal (∼ 50% carbohydrate, 30% fat, 20% protein, 6.2 kcal/kg body wt) with 250-ml of water or the test beverage (posttest). Posttest sessions were conducted at the same time of day as pretest sessions. Euhydration was achieved by instructing participants to drink water liberally the day before and to drink one 250-ml glass of water 1 h before testing. Upon arrival at the laboratory, participants completed a 24-h history form to assess compliance with pretest instructions, gave a urine sample (voiding all urine), and had tympanic temperature measured. Urine-specific gravity was measured with a refractometer to verify euhydration. Participants were not tested if urine-specific gravity was > 1.020, suggesting they were dehydrated (43), or if they had a fever (> 37.8°C). Next, maximal voluntary and electrically stimulated strengths of the participant's right knee extensor muscles were assessed. After that, nude body weight was measured, and a Teflon catheter was inserted into an antecubital vein. Participants mounted the cycle ergometer and remained at rest for at least 15 min to allow plasma volume to stabilize. Then, a blood sample was collected.
Following blood collection, participants cycled on the Lode stationary ergometer for 60 min at a power output estimated to elicit 50% V̇o2peak. During this phase, the ergometer was in the hyperbolic mode in which the power output was independent of pedal rate. Following minute 60, and a rest of 2–3 min during which time the ergometer was reprogrammed, participants were instructed to ride as hard as possible for 10 min, simulating an extended all-out effort at the end of a cycling race. During the all-out effort, the ergometer was in the linear mode in which an increase in pedaling speed increased the power output. The Lode Excalibur Sport default linear value of 0.042 was used for all participants. The work performed during the 10 min was used as a measure of performance. This type of preloaded test has been shown to have higher reliability than a test to exhaustion at a fixed work rate (24). During the 60-min ride, HR, RPE, and metabolic measurements were obtained during the last 3 min of every 15-min interval. Blood samples were obtained at minutes 15, 30, and 60 of cycling, as well as immediately following the 10-min performance ride. Following the cycling, participants toweled off and nude body weight was measured. Ad libitum fluid intake was permitted during the recovery. At 20 min following exercise, the measures of maximal voluntary and electrically stimulated strength of the subject's right knee extensor muscles were remeasured.
Maximal voluntary and electrically evoked isometric torques.
Similar to that previously described by us (10), isometric torque production of the right knee extensor muscles was assessed during both maximal voluntary contractions (MVC) and electrically evoked contractions (EEC). These measurements were made using a modified leg extension/curl machine (model NT-1220; Nautilus Fitness Products, Louisville, CO). The subject was placed in a semireclined seated position on the machine with hip and knee flexion angles set at 80° and 70°, respectively; these angles were determined from pilot work to be optimal for isometric torque production. The leg extension arm was attached to a force transducer (model SBO-300-T; Transducer Techniques, Temecula, CA), enabling the determination of isometric torque production about the knee. Using Velcro straps, the subject's right ankle was secured to the leg extension arm. Additionally, a seatbelt was strapped across the subject's waist and chest to prevent upper body movement. To enable electrical stimulation of the knee extensor muscles, two 8 × 10 cm adhesive electrodes (UniPatch 616SS; Wabasha, MN) were placed on the skin overlying the thigh, one each over the distal vastus medialis and proximal vastus lateralis muscles. Because the electrodes were removed for the cycling protocol, electrode position on the skin was marked with indelible ink to ensure that electrode positioning was the same after cycling as before.
Initially, a series of brief electrical stimulations (i.e., paired pulses, consisting of two 0.2-ms pulses with an interpulse interval of 10 ms) was delivered to the right knee extensor muscles while they were relaxed. Stimulations were administered using a constant current stimulator (model DS7AH; Digitimer, Hertfordshire, England) controlled by a 2.4 GHz Pentium computer using an A/D interface board (model KPCI-3116; Keithley Instruments, Cleveland, OH) and a custom program written with TestPoint software (version 6.0; Capital Equipment, Billerica, MA). This system also sampled the torque data at 5 kHz from the force transducer on the leg extension/curl machine. During the series of stimulations, the stimulator current was progressively increased until isometric torque production of the knee extensors had plateaued. Current was initially set at 100 mA and was increased by 20 mA every contraction. There was a 20-s interval between contractions. The current producing the greatest torque value within the plateau was determined to be the subject's supramaximal stimulation current and was used in the subsequent stimulations.
Next, the subject performed a 3-s isometric MVC of the knee extensor muscles and peak torque was recorded. At 2 and 4 s after the end of the MVC contraction (i.e., while the muscle was potentiated) the paired-pulse stimulation was administered to the relaxed muscle to determine the peak EEC torque; the average value for the two stimulations was used in subsequent analyses. Six sequential MVC/EEC measurements were made prior to cycling and also starting at 20 min after cessation of cycling. Within a sequence, there was 1 min of rest between the interpolated twitch procedures. For a given sequence, the three trials yielding the three highest MVC torques were determined and those data were averaged. Likewise, the three trials yielding the three highest EECs were determined, and those data were averaged.
Free (aglycone) and total plasma quercetin were determined by Gel Analytics (Golden, CO). Samples for the determination of quercetin aglycone were prepared by transferring 50 μl of plasma to a preconditioned Waters Oasis 96-well HLB μElution Plate. Then, 50 μl of acetate buffer (pH 5) containing the internal standard hesperitin was added to the plasma. The samples were extracted under a gentle vacuum followed by rinsing with 200 μl 95:5 H2O/MeOH. Quercetin was eluted with 200 μl 50:50 MTBE/MeOH, which was evaporated to dryness and reconstituted in 50 μl 0.1% formic acid.
Conjugated glucosides of quercetin were determined by enzymatic digestion prior to extraction. Samples were digested by combining 50 μl of plasma with 50 μl of enzyme solution containing 250 U/ml sulfatase (helix pomatia) and 5,000 U/ml β-glucuronidase (helix pomatia) and incubated for 2 h at 37°C. After incubation, samples were extracted as described above.
An Applied Biosystems 4000 QTrap mass spectrometer operating in the positive ion turbo spray mode was used for analysis. Separations were performed on a Waters Sunfire C18 4.6 mm × 50 mm, 2.5-μm column. The mobile phases were 0.1% FA/H2O and 0.1% FA/acetonitrile. The gradient was 25% of 0.1% FA/acetonitrile to 70% of 0.1% FA/acetonitrile in 5 min. Quantitation was accomplished using the ion transition m/z 303 to m/z 229 for quercetin and m/z 303 to m/z 177 for hesperitin.
Other blood measures.
Plasma volume change during cycling and recovery relative to preexercise rest were calculated from measures of hemoglobin and hematocrit (13). Hematocrit was measured using a microhematocrit centrifuge, and hemoglobin concentration was measured using a HemaCue analyzer. Plasma samples were assayed for glucose (Glucose LiquiColor Test procedure No. 1070; Stanbio Laboratory, Boerne, TX), nonesterified fatty acids [(NEFA)-HR assay kit; Wako Chemicals, Richmond, VA], glycerol (Sigma Free Glycerol Reagent cat. no. F6428) and β-hydroxybutyrate (liquicolor procedure No. 2440; Stanbio Laboratory, Boerne, TX).
Differences between groups Q and P in physical characteristics, body composition, and physical activity history were determined using t-tests for independent samples. Differences between groups Q and P in pretest-posttest changes on the outcome measures were analyzed using a two-way (group × treatment time) or three-way (group × treatment time × test time) mixed-model ANOVA with repeated measures for the factors involving time. The Geisser-Greenhouse correction was used when the sphericity assumption was violated.
Effect sizes (Cohen's d) were calculated as ΔMQ-ΔMP/SDPooled, where ΔMQ is the difference between posttest and pretest means of group Q, ΔMP is the difference between posttest and pretest means of group P, and SDPooled is the pooled SD from both groups before and after the intervention. The intervention did not substantially alter the SD in either group on any variable. By using a mixed-model repeated-measures ANOVA, a sample size of 15 per group is sufficient to detect a moderate group × time interaction effect of ∼0.55 standard deviation (SD) with an α of 0.05 and a power of 0.8, assuming a correlation between repeated trials of 0.9 (39). An effect equal to 0.55 SD would correspond to 4.6 s for PCr recovery rate, 229 ml/min or 3.0 ml·kg−1·min−1 for V̇o2peak, or 7.7 kJ of work on the performance trial, the primary outcome variables.
Participant characteristics and physical activity.
The physical characteristics, V̇o2peak and average daily energy expenditure of the participants in groups Q and P prior to the treatment were not different (Table 1). These data indicate that randomization created groups equivalent in physical characteristics, aerobic fitness, and physical activity. After the treatment, neither body weight nor physical activity, as measured by the 7-day physical activity recall, was significantly different (P > 0.05) compared with pretreatment values in either group.
Plasma quercetin was measured from blood samples taken at rest prior to the 60-min bout of cycling and cycling performance test. Prior to the treatment, neither free quercetin nor total quercetin was significantly different between groups Q and P. After the treatment, free quercetin changed little in either group (from 10.4 to 9.9 ηg/ml in group Q and from 9.9 to 10.6 ηg/ml in group P), although the group × treatment time interaction was statistically significant (P = 0.014). The changes in total quercetin by groups Q and P were significantly different (P < 0.001), increasing by 2.8-fold in group Q and decreasing slightly in group P (Fig. 1). These results demonstrate that ingestion of the experimental treatment beverage had the intended effect of elevating total plasma quercetin in group Q relative to group P.
V̇o2peak. Data on the effect of the treatment on V̇o2peak and related measures are presented in Table 2 and Fig. 2. The mean (± SD) of individual changes in V̇o2peak (l/min) was 87 ± 216 ml/min (2.7 ± 6.2%) or 1.0 ± 2.7 ml·kg−1·min−1 (2.4 ± 6.3%) in group Q and −11 ± 222 ml/min (−0.1 ± 6.7%) or −0.4 ± 2.4 ml·kg−1·min−1 (−0.7 ± 6.0%) in group P. The treatment effect size was small (d = 0.25). The mean change for time on the graded exercise test, a measure of work capacity, was 1.1 ± 2.2 min (8.5 ± 13.7%) in group Q and 0.6 ± 1.2 min (4.4 ± 9.2%) in group P. However, the group × treatment time interactions were not statistically significant (P > 0.05) for these measures, indicating that the pretreatment-to-posttreatment changes were not different in the two groups. The means for HR, respiratory exchange ratio, and RPE at V̇o2peak were typical of those expected, and indicated the participants in both groups gave a similar maximal effort at both tests.
Responses to cycling at 50% V̇o2peak.
Metabolic and perceptual responses assessed during the last 3 min of the 60 min of submaximal cycling at 50% V̇o2peak are presented in Table 3. There were no statistically significant differences (P > 0.05) in the pretreatment-to-posttreatment change for any of the metabolic, cardiorespiratory, or perceptual measures. However, there was a tendency for the decrease in V̇o2 to be greater in group Q than in group P. Similarly, the respiratory quotient (RQ), reflecting the percentage of energy obtained from the oxidation of carbohydrate and fat increased slightly in both groups, and the change in RQ in group Q was almost significantly greater than the change in group P. RPE tended to decrease more in group Q than in group P, although the differences were not statistically significant.
Plasma volume decreased 2–4% in groups Q and P during the first 15 min of the ride, and by 60 min was 1–2% below rest. After the 10-min performance trial, plasma volume was reduced 10–12%. In the three-way (group × treatment time × test time) ANOVA, neither the three-way interaction (P = 0.086) nor the group × treatment time interaction (P = 0.82) was significant, indicating that the pretreatment-to-posttreatment changes by the groups were not different.
Plasma concentrations of glucose, NEFA, glycerol, or β-hydroxybutyrate during the 60-min ride and following the 10-min performance trial before and after the treatment are shown in Fig. 3. There were no statistically significant group × treatment time × test time or group × treatment time interactions, indicating that pretreatment-to-posttreatment changes in the two groups did not differ. These data taken together with the RQ results indicate that substrate mobilization and utilization during exercise were unchanged by the treatment.
Adequate PCr depletion and recovery curves with good curve fits were obtained on 11 participants in group Q and 13 participants in group P. An example of PCr changes during exercise and recovery is shown in Fig. 4. Quadriceps isometric exercise before and after treatment depleted PCr an average of 38 ± 13% and 38 ± 9% in group Q, and 33 ± 10% and 39 ± 11% in group P. At rest and at the end of exercise, pH averaged 7.00 ± 0.06. During recovery, the minimum pH averaged 6.80 ± 0.09 in both groups before and after the treatment. These data indicate that the exercise protocol did not elicit pH changes of the magnitude that affect the kinetics of PCr recovery (47). Although the means of individual PCr recovery time constants (τ) increased in groups Q and P from pretreatment to posttreatment, in the two-way ANOVA, the group × treatment time interaction was not statistically significant (P = 0.64), indicating there was no difference between the groups in the pretreatment-to-posttreatment change in PCr recovery τ (Fig. 5). The treatment effect size was low (d = 0.13).
Cycling performance, as measured by the work performed during the 10-min maximal effort cycling trial following the 1 h of cycling at 50% V̇o2peak was not affected by the treatment. The mean (± SD) change in total work performed from pretreatment to posttreatment was 3.0 ± 5.3 kJ (2.7 ± 4.4%) in group Q and −0.7 ± 12.2 kJ (−0.09 ± 9.6%) in group P (Fig. 6). The treatment effect size was low (d = 0.21). The group × treatment time interaction was not statistically significant (P > 0.05), however, indicating that the pretreatment-to-posttreatment changes were not different in the two groups. Similarly, the group × treatment time interaction for the V̇o2 during the last minute of cycling during the performance trial, which should be proportional to the work performed, was not statistically significant (P > 0.05). Mean values for V̇o2, HR, and RPE in participants in groups Q and P before and after the treatment averaged 89–98% of values observed at the point of fatigue at the end of the graded exercise test (Table 4), indicating a good effort was given by both groups on the performance trials.
Voluntary isometric knee-extension strength decreased an average of 32 ± 9 kg (10 ± 3%) in group Q and 14 ± 12 kg (5 ± 3%) in group P during the 60 min of cycling and subsequent performance trial during the pretreatment tests and 20 ± 7 kg (6 ± 2%) in group Q and 29 ± 9 kg (7 ± 2%) in group P during the posttreatment tests. The group × treatment time interaction for voluntary strength loss was not statistically significant (P = 0.17), indicating that there was no effect of the treatment on voluntary strength loss during exercise. Likewise, electrically evoked isometric strength decreased an average of 24 ± 2 kg (16 ± 2%) in group Q and 28 ± 5 kg (16 ± 2%) in group P during the 60 min of cycling and subsequent performance trial during the pretreatment tests and 18 ± 3 kg (11 ± 2%) in group Q and 14 ± 4 kg (7 ± 3%) in group P during the posttreatment tests, but the group × treatment time interaction was not statistically significant (P = 0.33), indicating that there was no effect of the treatment on electrically evoked strength loss during exercise.
The primary purpose of our study was to determine whether dietary supplementation with a beverage containing quercetin improves skeletal muscle oxidative capacity and has other metabolic, neuromuscular, and neural consequences that improve performance in prolonged exercise in untrained men. Our primary findings are that 7–16 days of dietary quercetin supplementation (1 g/day) did not affect muscle oxidative capacity; measures of substrate utilization, cycling economy, or perception of effort during constant rate submaximal exercise; prolonged cycling performance; or strength loss following cycling in untrained men. Our findings do not support the hypothesis that quercetin has ergogenic effects. The null findings are important because they indicate that metabolic and physical performance consequences of quercetin supplementation observed in mice should not be generalized to humans.
Quercetin supplementation with 1 g/day for 9–16 days in our study did not enhance performance in prolonged exhaustive cycling. Work performed during the 10-min performance trial following 60-min of submaximal cycling at 50% V̇o2peak did not increase significantly more in group Q than in group P. Approximately the same number of participants in group Q had poorer performance as had better performance. Our results are consistent with the few published studies on the ergogenic effect of quercetin supplementation in humans. MacRae and Mefferd (29) found that 6 wk of dietary supplementation with an antioxidant health drink containing 600 mg/day of quercetin did not improve performance on a 30-km cycling time trial significantly more than supplementation with the cocktail without quercetin (placebo). Nieman et al. (36) reported that ultramarathoners competing in a 160-km run randomly assigned to quercetin (1 g/day) and placebo treatments for 3 wk prior to and on the morning of the race did not differ in race times. Similarly, 2 wk of quercetin supplementation (1 g/day) failed to increase cycling time trial performance of cyclists during 3 days of intensified training (37). Cheuvront et al. (9) found that acute quercetin supplementation (2 g) did not alter work performed in a preloaded cycling time trial in the heat.
There are several mechanisms through which quercetin supplementation might improve performance during prolonged, exhaustive exercise. One mechanism would be through an increase in skeletal muscle oxidative capacity due to mitochondrial biogenesis. Studies on sedentary mice found that quercetin (11) and a related polyphenol, resveratrol (28), stimulate mitochondrial biogenesis, increasing skeletal muscle mitochondrial protein, oxidative enzyme activity, and endurance running performance. These polyphenols appear to activate sirtuin 1 and PGC-1α, which in turn activate transcription factors leading to mitochondrial biogenesis (28). Transgenic mice that have upregulated PGC-1α have increased skeletal muscle oxidative capacity, peak oxygen consumption, and running performance (8).
We used the time constant for rate of PCr recovery (τ) following brief exercise measured with 31P-NMR spectroscopy to assess any change in skeletal muscle oxidative capacity, as done previously (32). PCr recovery rate and citrate synthase activity measured in the gastrocnemius muscles correlate ∼0.7–0.8 in animals and humans (31, 38). Endurance training of the calf muscles systematically decreased the PCr recovery τ by ∼20% over 2 wk (32) and six sessions of sprint training over 2 wk decreased PCr recovery τ in the quadriceps muscles by 14% (15). PCr recovery τ values vary from ∼18 s in endurance-trained athletes to over 40 s or longer in sprinters, the elderly, sedentary, and diseased individuals (35, 36, 38).
The PCr recovery τ means for our participants in groups Q (33 s) and P (30 s) prior to the treatment were similar to those of young, non-endurance-trained controls in other studies (31, 32). Although PCr recovery τ increased for an unknown reason in both groups, there was no difference between the groups in the change. These data indicate that quercetin did not cause a substantial (20–30%) increase in muscle oxidative capacity in group Q compared with group P, such as the changes reported to result from quercetin (11) or resveratrol (28) feedings in mice. Failure to detect a difference between groups could be due to insensitivity of the measurement, lack of an effect, or inadequate statistical power. The sensitivity of the measurement should have been able to detect a change of 20–30% in muscle oxidative capacity, as shown previously with the increased skeletal oxidative capacity resulting from exercise training (15, 32). Changes in PCr recovery τ in groups Q and P from pretreatment to posttreatment were similar, with no evident effect in the hypothesized direction. Inadequate statistical power was not a problem, because there was no effect. Our findings are consistent with those of Nieman et al. (37), who reported 2 wk of quercetin supplementation at 1 g/day did not affect muscle oxidative capacity in trained cyclists. It is not known whether high values for muscle oxidative capacity in the trained cyclists prevented an additional increase due to quercetin, but such a ceiling effect could be hypothesized from the absence of mitochondrial biogenesis in heart muscle with high oxidative capacity in sedentary mice in whom skeletal muscle oxidative capacity was increased by resveratrol (28).
If muscle oxidative capacity had increased substantially as in the study of Davis et al. (11) in mice, an increase in V̇o2peak would be expected through an increase in oxygen extraction and the arteriovenous oxygen content difference. An increase in muscle oxidative capacity can increase V̇o2peak independent of any change in blood flow to the active muscles (30). Thus, although V̇o2peak is thought to be limited by the capacity of the heart to increase cardiac output and oxygen delivery (4), quercetin supplementation could theoretically increase V̇o2peak through effects on skeletal muscle alone (19). This deduction is supported by the increased V̇o2peak in sedentary transgenic mice who increased skeletal muscle oxidative capacity and V̇o2peak, due to upregulated PGC-1α.
Consistent with our finding of no change in muscle oxidative capacity, quercetin supplementation did not significantly increase V̇o2peak. V̇o2peak increased an average of 87 ml/min (2.7%) in group Q and decreased 11 ml/min (−0.1%) in group P following the supplementation period. The difference between the two groups in changes in V̇o2peak was not statistically significant. The magnitude of the treatment effect was small and less than the sum of the technical error and normal biological variability in V̇o2peak (∼5%) (26). Although it is possible that quercetin had a small effect that was not detected due to inadequate statistical power, the biological and practical importance of a change less than the normal biological variability in an individual is limited. Our data indicate that either there is no effect or only a very small (∼1 ml·kg−1·min−1, 3%) effect of 7–9 days of 1 g/day quercetin supplementation on V̇o2peak.
Another mechanism through which quercetin supplementation could improve performance in prolonged exercise is by altering substrate utilization to spare muscle glycogen and glucose, although this was not likely to be a factor of major importance in our study due to the relatively short duration of the preloaded cycling test (∼70 min). Increased mitochondrial density and muscle oxidative capacity resulting from endurance exercise training alters substrate utilization by increasing the reliance on fat for fuel and reducing reliance on muscle glycogen and glucose (20). Quercetin also may increase fat mobilization via effects on adenosine receptors on adipocytes (27). Increased fat utilization might also be expected if V̇o2peak is increased and a given submaximal exercise intensity requires a lower percentage of V̇o2peak. These changes can delay fatigue and improve performance during prolonged exercise by increasing carbohydrate availability (20). Consistent with lack of changes in muscle oxidative capacity and V̇o2peak, we did not find that quercetin supplementation altered substrate utilization. The pretreatment-to-posttreatment changes in RQ and plasma concentrations of glucose, NEFA, glycerol, and hydroxybuterate during constant rate submaximal exercise were not different in groups Q and P. Our findings do not support the suggestion that quercetin might increase performance in prolonged exercise altering substrate utilization.
It has been hypothesized that the antioxidant properties of quercetin may improve exercise performance by minimizing damage to membranes and contractile and structural proteins in skeletal muscle, thereby limiting the acute negative, fatiguing effects of increased reactive oxygen species generated during exercise (29). If this is true, antioxidant supplementation might reduce muscle damage, soreness, and impaired neuromuscular function observed after exercise. We found quercetin supplementation did not affect neuromuscular function, as reflected by voluntary and electrically evoked strength loss following 1 h of cycling and the 10-min time trial to exhaustion. The fact that electrically evoked and voluntary strength losses following exercise were unaffected by quercetin indicates that quercetin did not invoke neural or muscle changes responsible for the strength loss. Our findings do not support the suggestion that the antioxidant properties of quercetin might augment performance in prolonged exercise by reducing loss in neuromuscular performance.
Another mechanism through which cycling performance might theoretically have been improved by quercetin supplementation is by decreasing perception of effort or pain, as occurs with caffeine (10, 14, 35). Quercetin has been shown in in vitro tests to be an adenosine A1-receptor antagonist (1) and thus may have analgesic effects and direct effects on muscle, augmenting force-producing capability (23). We did not find an effect of quercetin supplementation on RPE during the 1 h of steady-state cycling prior to the performance test. Our findings on untrained men are consistent with a study on chronic ingestion of quercetin in competitive runners (46) and one study on acute ingestion of quercetin in moderately fit men studied during cycling in the heat (9). The lack of an effect of quercetin on RPE may have been because the plasma total quercetin concentration was not increased enough. The inhibition constant for the adenosine receptor occurs at a quercetin concentration > 700 ng/ml (25). Our plasma quercetin levels were much lower than this and, therefore, may not have been high enough to produce an effect. However, levels of plasma quercetin much higher than 700 ng/ml have not altered RPE during submaximal exercise (9). Taken together, our findings and those of others suggest RPE during prolonged exercise are not reduced by chronic dietary supplementation or by acute ingestion of quercetin. Our findings do not support the suggestion that quercetin might increase performance in prolonged exercise by decreasing RPE as has been reported for caffeine (10, 14, 35).
The results of our study suggest that quercetin does not increase muscle oxidative capacity and improve performance in prolonged exercise in untrained humans as it does in mice that were administered a comparable dose (mg/kg body wt) of quercetin for a similar length of time (11). Also, other effects that would be expected, if muscle oxidative capacity increased, that mimic adaptations to endurance exercise training (20), such as increased V̇o2peak and altered substrate utilization, do not appear to occur. We hypothesized that failure to find increased muscle oxidative capacity (37) or improved performance in prolonged exercise (29, 36, 37) following quercetin supplementation in previous studies on highly trained athletes may have been because the athletes had reached a ceiling for increasing muscle oxidative capacity due to high levels attained through exercise training. Lack of ability to increase muscle oxidative capacity should not have been a problem in the current study. Young, healthy, non-endurance-trained men were used as participants. All were college students who engaged in light and moderate physical activity in carrying out daily tasks and participated in recreational activities, but none participated in sustained aerobic exercise > 20 min, 1 day/wk. Data from the 7-day recall of physical activity confirmed that the participants could be categorized as relatively inactive based on their average daily physical activity (6). Other data indicated that the participants were unfit. Mean values for V̇o2peak (ml·kg−1·min−1) were below average, and those for percentage of fat were above average, compared with young men of their age (3). In this sample of relatively inactive, low-fit men, mitochondrial density and oxidative enzyme activities should average approximately one-half values reported for highly trained endurance athletes (18, 29), such as the cyclists and runners studied in previous reports of the effects of quercetin on muscle oxidative capacity, and endurance performance (29, 36, 37). Thus, in this sample, there was ample opportunity for adaptations in muscle oxidative capacity and V̇o2peak that would improve performance during prolonged, exhaustive exercise.
It is possible that the effective dose/duration of quercetin needed to cause, or steps and/or time course involved in, mitochondrial biogenesis in humans are different than in mice. The dose of quercetin (ml·kg−1·day−1) and duration of treatment in our study was patterned after the study of Davis et al. (11) on mice. They found that quercetin supplementation with 12.5 ml·kg−1·day−1 or 25 mg·kg−1·day−1 for 7 days increased skeletal muscle cytochrome c concentration 18–32%, and treadmill run time to fatigue by 36–37%. No comparable data exist on humans providing insight into the dose or duration of quercetin supplementation needed to stimulate mitochondrial biogenesis, if this adaptation occurs. The dose equivalent to 12.5 mg quercetin/kg body wt in humans, assuming a body weight of 70 kg, is 875 mg/day. The dose in the current study (1 g/day) was slightly above this level. It is possible this dose is too high, because the recommended basis of translation of animal to human doses is based on body surface area rather than body weight (41). Using the recommended conversion of mouse to human dosages (mouse dose * 0.08), the human dose equivalent to 12.5 mg·kg−1·day−1 would be 1.01 mg·kg−1·day−1 or, for a 70-kg man, 71 mg/day. It is also possible that a lower or higher dose in humans is needed because the bioavailability, metabolism, or tissue distribution of quercetin is different in humans and mice (12).
Our failure to find increased muscle oxidative capacity, V̇o2peak, or cycling performance may have been because of an inadequate treatment duration. Although some participants in group Q received the quercetin treatment for a longer period of time prior to the performance test than others (9–16 days), there was no relation between duration of treatment and performance change. Although Davis et al. (11) reported that a relatively large increase in skeletal muscle oxidative capacity occurred with 7 days of quercetin feedings in mice, a similar adaptation may require a longer supplementation in humans. Interspecies differences in quercetin bioavailability, metabolism, and partitioning among tissues may explain the inconsistency in the effects of quercetin on different species. Because quercetin activates sirtuin 1 and PGC-1α (11), at least some of the cell signaling steps involved in initiation of mitochondrial biogenesis appear to be similar to those in muscle contraction-induced mitochondrial biogenesis (22). In humans and other mammals, the initial cell signaling events are initiated following acute bouts of exercise, with protein accumulation and assembly into complexes and phospholipid content changes accumulating over time. An increase in the steady-state level of mitochondria, muscle oxidative capacity, and V̇o2peak typically requires ∼6 wk of exercise training (18, 21), although the adaptation to a continuously elevated level of quercetin could be more rapid. Thus, it is possible that mitochondrial adaptations occur with quercetin supplementation in humans, but with a different dose of quercetin and/or duration of treatment than in mice.
Failure to find an ergogenic effect of quercetin could have been due to an interaction of quercetin with other ingredients in the beverage that reduced its bioavailability or biological activity. If we had found a positive effect of quercetin, we would have been unable to determine whether the effect was due to quercetin or to an interaction of quercetin with another beverage ingredient. Future studies on the efficacy of quercetin should evaluate the effects of possible interactions.
Finally, our null findings may have been due to the negative effects of antioxidant properties of quercetin on PGC-1α, PPARγ, and mitochondrial biogenesis. Ristow et al. (42) have shown that the anitoxidant vitamins C and E block exercise-training-induced increases in PGC-1α, PPARγ, regulators of mitochondrial biogenesis, and markers of insulin sensitivity in previously trained and untrained individuals.
We conclude that short duration, chronic quercetin supplementation does not improve muscle oxidative capacity; metabolic, neuromuscular, or perceptual determinants of performance in prolonged exercise; or cycling performance in untrained men. The null findings indicate that metabolic and physical performance consequences of quercetin supplementation observed in mice should not be generalized to humans.
This research was supported by The Coca-Cola Company.
The authors thank Sahir Ahsan, Scott Cole, Drew Dixon, Sequioa duCasse, Kim Mason, Robert Moore, Gordon Warren, and Paul Winkler for their technical and professional assistance with the study.
- Copyright © 2009 the American Physiological Society