Introduction
Caffeine (CAF) is one of the most widely used drugs in the world. In moderate doses, CAF is well tolerated, and few significant side effects have been reported (10,42). Several studies have demonstrated improvements in cycling performance after the administration of CAF in moderate to high doses (3–13 mg·kg−1 of body mass [BM]) (2,3,9,12,18,19,21,24,30,33,34,39). Further, the administration of CAF in lower doses (<3 mg·kg−1 BM) has been shown to enhance cycling performance (9,25,30). Relatively few studies reported that CAF (3–6 mg·kg−1 BM) had no effect on cycling performance (7,13,23).
The pharmacokinetics of CAF are well understood. After ingestion, CAF is rapidly and completely absorbed from the gastrointestinal tract into the bloodstream (17). Complete absorption is typically achieved approximately 1 hour after ingestion; however, numerous factors such as dosage and dose formulation can impact the absorption rate constant (5). After the oral ingestion of CAF, plasma CAF concentrations increase in a dose-dependent manner (27), and peak plasma concentrations are observed anywhere from 15 to 120 minutes (4,5,28,29,32,37). As such, it has been customary for researchers to administer oral doses of CAF (capsule or beverage) 1 hour before performance to ensure peak plasma concentrations during exercise.
Research conducted by Kamimori et al. (27) demonstrated that CAF administered in chewing gum is absorbed significantly faster than CAF administered in pill form. Because drug action is limited by the rate at which a drug reaches target tissues, a faster delivery of CAF may result in a faster onset of ergogenic effects. Nonetheless, research has not elucidated the temporal relationship between the timing of administration of CAF gum and the subsequent enhancement of cycling performance. We hypothesized that the administration of CAF in chewing gum would elicit an ergogenic effect very rapidly. The purpose of this study was to determine the most efficacious time to administer CAF in chewing gum to enhance cycling performance.
Methods
Experimental Approach to the Problem
A within-subject, repeated measures, placebo controlled design was chosen for this study with all the subjects being tested under all treatment conditions. The subjects participated in 4 experimental trials in which cycling performance was assessed via a cycling time trial. Caffeine was administered at one time point in 3 trials and placebo administered at all time points in a control trial to allow for comparisons between trials. Stay Alert chewing gum (Mastix Medica LLC, Hunt Valley, MD, USA) was used in the current investigation as previous studies conducted to determine the pharmacokinetics of CAF administered in chewing gum used this product (27,43). To control for possible sources of error variance, CAF was administered in a double blind manner, and the order in which subjects completed the trials was randomized.
Subjects
Eight college-aged (25 ± 5 years), trained (50 ± 5 ml·kg−1·min−1) male cyclists were recruited via direct contact with the principal investigator. Volunteers were non-to-moderate CAF users (<300 mg·d−1) who were not taking medication or using dietary supplements of any kind. The majority of subjects were CAF naive, whereas 2 reported CAF use of <2 cups of coffee a day. All the subjects reported participating in both aerobic and strength training routines. They were asked not to alter their training habits throughout their participation in the study (testing completed within 1 month). Informed consent was obtained from each subject before participation, and this protocol was approved by the Kent State University Institutional Review Board for the Protection of Human Subjects.
Procedures
Preexperimental Testing
The participants reported to the Exercise Science Laboratory at which time height and BM measurements were obtained via a stadiometer and a balance beam scale, respectively. The participants then underwent a graded exercise test on a Lode cycle ergometer (Groningen, The Netherlands) until volitional fatigue. To determine maximal oxygen consumption (V[Combining Dot Above]O2max), air samples were analyzed for oxygen and carbon dioxide concentrations via an indirect open circuit spirometry system (Parvomedics, Metabolic Cart, Sandy, UT, USA).
Experimental Testing
Figure 1 provides an overview of the experimental protocol. The subjects participated in 4 experimental trials in which cycling performance was assessed via a 7-kJ·kg−1 cycling time trial. During the experimental trials, 3 pieces of chewing gum were administered at 3 time points (120-minute precycling, 60-minute precycling, and 5-minute precycling). The subjects were instructed to chew the gum for 5 minutes and then expectorate the gum. In 3 of the 4 visits, at 1 of the time points mentioned previously, 300 mg of CAF was administered. During the fourth visit, placebo gum was administered at all 3 time points. Stay Alert chewing gum (Mastix Medica LLC, Hunt Valley, MD, USA) was used in the current investigation because previous studies conducted to determine the pharmacokinetics of CAF administered in chewing gum used this product (27,43).
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Figure 1: Overview of experimental protocol. WU = warm-up; CAF = caffeinated gum; PLA = placebo gum; VO2 = oxygen consumption; RER = respiratory exchange ratio; HR = heart rate; RPE = ratings of perceived exertion; LP = perceived leg pain; blood = venous blood sampling.
One piece of Stay Alert chewing gum (Mastix Medica LLC) contains 100 mg of CAF, and it has been reported that 85% of the dose is released and absorbed during the first 5 minutes of chewing (27). Thus, it was anticipated that the subjects ingested approximately 255 mg of CAF. The CAF and placebo gum were virtually identical in shape, color, texture, and taste. The experimental trials are defined as follows: trial A (–120), trial B (–60), trial C (–5), and trial D (Placebo).
All the experimental trials were held between 0600 and 1200 and were conducted in the late winter months. Although all the subjects reported participating in strength training and aerobic endurance training, they were asked not to alter their training habits throughout the duration of their participation (within 1 month). Furthermore, the participants were requested to refrain from exercise and caffeinated or alcoholic beverages for at least 48 hours before the experimental trials. The subjects reported to the laboratory in the postabsorptive state. To prevent dehydration, the subjects were permitted to drink water at liberty throughout experimental testing.
Upon arrival at 0600, the subjects were weighed via a balance beam scale. The subjects then consumed a light breakfast (370 cal, 2 g fat, 77 g carbohydrates, 11 g protein, 28 g sugar) consisting of a plain bagel and 240 ml of orange juice. After breakfast, baseline expired air samples and heart rate (HR) values were obtained via an indirect open circuit spirometry system (Parvomedics, Metabolic Cart) and a Polar HR monitor (Accurex Plus, Polar Electro, Inc., Woodbury, NY, USA), respectively. Venous blood sampling was then performed via an antecubital venipuncture. After baseline measurements, chewing gum was administered at the aforementioned time points. Between administration time points, the participants rested in the seated position and were allowed to read, converse with data collectors, use the restroom, and use technology. Ten minutes before steady-state cycling an additional venous blood sample was obtained. The subjects then completed a standard warm-up consisting of cycling at a desired workload.
Steady-State Cycling
All cycling was performed on a Lode cycle ergometer (Groningen, The Netherlands). After time allotted for baseline measurements and gum administration and a standard warm-up, the participants cycled at constant wattage (workload corresponding to 75% V[Combining Dot Above]O2max) for 15 minutes. During steady-state cycling, metabolic and perceptual measurements were obtained to allow for comparison between trials. Expired air samples were collected and analyzed for oxygen and carbon dioxide concentrations throughout steady-state cycling. The HR was measured via a Polar HR monitor, and HR values were recorded every 3 minutes. Ratings of perceived exertion (RPE) recordings were obtained every 3 minutes via Borg's RPE 6–20 scale (6). Perceived leg pain (LP) was assessed every 3 minutes via a 0–10 pain sensation scale (8).
Cycling Time Trial
After steady-state cycling, venous blood sampling, and a 5-minute active cool-down, cycling performance was assessed via a cycling time trial. The cycle was set on linear mode (RPM dependent) and a linear factor was calculated for each subject based on the following equation: W = L × (RPM)2. The linear factor was determined in such a way that when the subjects cycled at a preferred RPM a workload corresponding to 75% V[Combining Dot Above]O2max was achieved. The participants were instructed to perform a set amount of work (7 kJ·kg−1) as quickly as possible. During the cycling time trial, the subjects only viewed work achieved and were not informed of exercise time. This type of performance measure has been shown to have high reproducibility and a low coefficient of variation for the outcome measure of performance time (26).
Blood Sampling and Analyses
Venous blood samples were obtained at 4 time points: 125-minute precycling, 10-minute precycling, immediately after steady-state cycling, and immediately after the completion of the cycling time trial. Venous blood samples were drawn from the antecubital space via venipuncture and collected. The purpose of the blood draws was to obtain plasma β-endorphin concentrations, plasma CAF concentrations, and hematocrit and hemoglobin measurements to determine plasma volume shifts (14).
Samples collected in green top tubes containing sodium heparin were immediately centrifuged for 5 minutes at 3,700 rpm (4° C), and aliquoted. Plasma samples were stored at –80° C and later analyzed for CAF concentrations using a modified high-performance liquid chromatography method (41). Briefly, 250 μl of 0.8 M perchloric acid containing 25 μg·ml−1 of the internal standard 8-chlorotheophylline was added to 250 μl of plasma. The resulting solution was vortexed for 5 seconds, then centrifuged at 6,000 rpm for 5 minutes. Fifty microliters of the supernatant was injected into the chromatographic system (Agilent 1100 series). Caffeine was eluted with a Waters Resolve C18 (3.9 × 150 mm) analytical column. The mobile phase consisted of KH2PO4/acetonitrile/tetrahydrofuran (88:9.2:2.8 v:v:v) and was pumped at 1 ml·min−1. Eluted peaks were detected using ultraviolet absorption at a wavelength of 274 nm, and peak areas were used for quantification using a 5-point standard curve. The limit of quantification was 500 ng·ml−1.
Samples collected in 7-ml lavender (ethylenediaminetetraacetic acid [EDTA]) tubes were immediately transferred into centrifuge tubes containing aprotinin (0.6 TIU per milliliter of blood) and gently rocked several times to inhibit the activity of proteinases. The samples were then centrifuged for 15 minutes at 1,600 rpm (4° C) and aliquoted. Plasma samples were stored at −80° C and later analyzed for β-endorphin concentrations via a valid, commercially available enzyme immunoassay (β-endorphin enzyme-linked immunosorbent assay, M056011, MD Biosciences Inc., St. Paul, MN, USA) (1,40). Samples collected in 3-ml lavender (EDTA) tubes were immediately analyzed for hematocrit and hemoglobin concentrations. Hematocrits were measured in triplicate in a microhematocrit centrifuge (Critspin, Iris sample processing, Westwood, MA, USA), and hemoglobin was measured via the cyanmethemoglobin method (Eagle hemoglobin procedure, Cima Scientific, Dallas, TX, USA). After the aforementioned analysis and collection of the resulting data, the blood samples were placed in a biohazard material container and disposed of by Kent State University.
Statistical Analyses
All data were analyzed via SPSS 16.0 software. To identify differences in cycling performance between experimental treatments (trial A [–120], trial B [–60], trial C [–5], and trial D [Placebo]), cycle time data were assessed using a repeated measures analysis of variance (ANOVA). To determine if CAF gum impacted metabolic and perceptual responses to exercise, data were analyzed using a 4 (treatment) by 6 (time) ANOVA with repeated measures on both variables. To determine significant differences between treatments in blood concentrations of CAF throughout the experimental trials, plasma, CAF data were analyzed using a 4 (treatment) by 4 (time) ANOVA with repeated measures on both variables. In the event that an ANOVA revealed a significant main effect or interaction, further exploration via paired samples t-tests with Bonferroni adjustments was performed. To determine if CAF influenced β-endorphin responses to exercise, β-endorphin data were evaluated using a mixed effects model.
Results
Data are presented as mean ± SEM. A main effect of treatment was observed for cycling time trial performance (p = 0.027) (Table 1). Further exploration showed that cycling performance was enhanced in trial C (–5) (p = 0.023) but not in trial A (–120) or trial B (–60), vs. trial D (Placebo). Plasma CAF concentration data are presented in Figure 2. Plasma CAF concentration demonstrated a treatment by time interaction (p < 0.001). Interestingly, plasma CAF concentrations did not differ between CAF trials after steady-state cycling (p ≥ 0.141) or after the cycling time trial (p ≥ 0.568). Although, plasma CAF concentrations continued to increase during the cycling time trial in trial C (–5) (p = 0.046), but not in trial A (–120) (p = 0.514) or trial B (–60) (p = 0.831).
Table 1: Cycle time trial performance, M ± SEM.
Figure 2: Plasma caffeine concentrations at baseline (Base), 10-minute precycling (−10), after steady-state cycling (+15), and after the cycle time trial (Post) across experimental treatments.
As expected, main effects of time were observed for oxygen consumption, respiratory exchange ratio, HR, RPE, LP, and plasma β-endorphin concentrations (p ≤ 0.035). However, no main effects of treatment or treatment by time interactions were evident for any of these variables (p ≥ 0.253).
Discussion
Caffeine is a socially acceptable drug that is readily available for athletes to use for performance enhancement both in competition and during training sessions. The most efficacious time to administer CAF in any formulation to maximize its performance enhancing effects is poorly understood (7). In the current investigation, cycling performance improved when an expected dose of 255 mg (mean relative dose of 3 mg·kg−1 BM) of CAF was administered in chewing gum immediately before cycling vs. placebo. Conversely, cycling performance was not enhanced when CAF gum was administered 1 or 2 hours before cycling. These data demonstrate that 3 mg·kg−1 BM of CAF administered in chewing gum enhances cycling performance when ingested immediately before exercise.
Blood analysis showed that plasma CAF concentrations did not differ between the 3 CAF trials immediately before and after performance testing. Moreover, plasma CAF concentrations continued to rise during the cycling time trial in trial C (–5) but remained stable during the cycling time trial in trial A (–120) and trial B (–60). These data suggest that the ergogenic effect of CAF was not evident when CAF gum was administered 1 or 2 hours before exercise even though plasma CAF levels were maintained. Furthermore, these data demonstrated that performance enhancement with CAF was evident only when plasma CAF levels were still rising during the cycling time trial.
Few studies have investigated the duration of the performance enhancing effect of CAF. In this study, cycling performance was not enhanced when CAF gum was administered 1 or 2 hours before cycling. Contrary to the present findings, Bell and McLellan (2) reported that CAF improves cycling performance in users at, 1 and 3, and nonusers at 1, 3, and 6 hours after ingestion. These equivocal findings can be explained by methodological differences. Bell and McLellan (2) had subjects ingest a larger dose (5 mg·kg−1 BM) of CAF than was used here. Also, in this study, CAF was administered in chewing gum, whereas in the study conducted by Bell and McLellan, CAF was administered in capsule form.
Researchers have reported that CAF ingestion decreases RPE (11,15,16,22) and LP (20,35,36,38) during cycling exercise. In this study, CAF did not impact RPE and LP during steady-state cycling; although, performance enhancement with CAF was evident. Jenkins et al. (25) also reported that CAF improved cycling performance but did not decrease RPE and LP during steady-state cycling. The subjects in the study conducted by Jenkins et al. (25) consumed 3 mg·kg−1 BM of CAF, then cycled 15 minutes at 80% V[Combining Dot Above]O2max. The participants in the current investigation consumed a mean relative dose of CAF equivalent to 3 mg·kg−1 BM, then cycled 15 minutes at 75% V[Combining Dot Above]O2max. Based on these findings, it is concluded that a CAF dose in excess of 3 mg·kg−1 BM may be necessary to reduce perceived exertion and pain during cycling exercise.
As expected, plasma β-endorphin concentrations increased with exercise in this study. However, CAF had no effect on plasma β-endorphin concentrations at rest and immediately after cycling exercise. Contrary to these findings, Laurent et al. (31) reported that CAF increased β-endorphin concentrations after 2 hours of cycling at 65% V[Combining Dot Above]O2max. One likely explanation for these equivocal findings is that a much larger dose of CAF (6 mg·kg−1 BM) was administered in that study relative to this study.
We acknowledge that this study has some limitations. Numerous factors such as dosage, dose formulation, and route of administration can impact the pharmacokinetics of CAF (5,27). Thus, the results presented here are specific to CAF (3 mg·kg−1 BM) administered in chewing gum. Future research is necessary to determine the effect of CAF administration timing on cycling performance when CAF is administered in larger doses and other formulations. Further, not accounting for dietary intake and sleeping habits may present possible sources of error variance.
In conclusion, CAF (3 mg·kg−1 BM) administered in chewing gum improved cycling performance when administered immediately before a bout of cycling exercise lasting approximately 1 hour. Plasma CAF concentrations were comparable between all CAF treatments before and after performance testing; however, cycling performance was not enhanced with CAF when the gum was administered at 1 or 2 hours before cycling
Practical Applications
We conclude that CAF gum should be administered immediately before exercise to enhance cycling performance for an event lasting approximately 1 hour. Furthermore, administering CAF gum ≥1 hour before an event lasting 1 hour does not enhance cycling performance. These findings are not inferable to cycling performance when CAF is administered in other formulations. However, based on these results, it is recommended that researchers administer CAF in a manner that allows for an increase in plasma CAF concentrations during performance to obtain the best ergogenic effect.
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