A total of 36 male recreationally competitive cyclists participated in the present study. One of these participants was excluded from the study post-hoc, as their cycling performance differed by more than two standard deviations from the mean value of the group. Therefore, 35 cyclists (age = 25.0 ± 7.3 yrs, height = 178.2 ± 8.8 cm, weight = 74.3 ± 8.8 kg, VO2max = 59.35 ± 9.72 ml·kg-1·min-1) were used for data analysis. Written informed consent was obtained from all participants prior to participation and the study and consent form were approved by the James Madison University Institutional Review Board. Habitual caffeine intake was self-reported by participants. Briefly, participants were asked for their average weekly intake of coffee, tea, soda, chocolate, and other caffeinated beverages. Typical milligram doses  were assigned to each and an approximate daily intake was obtained. Based on previous criteria , participants were then characterized as having low (0-150 mg·day-1), moderate (151-300 mg·day-1) and high (> 300 mg·day-1) caffeine intake.
Maximal exercise test
Cyclists began the test at a work rate of 150 W on an electrically braked cycle ergometer, with load increases of 20 W each minute until volitional exhaustion. Maximal oxygen uptake (VO2max) was defined as the highest 1-minute oxygen value obtained during the test. Oxygen uptake (VO2) was monitored continuously via a Sensormedics Vmax (Yorba Linda, CA) metabolic measurement system calibrated in advance of all tests. Heart rate was monitored throughout the test using a Polar Heart Rate Monitor (Lake Success, NY).
40-kilometer time trial
Time trials were performed on two separate occasions. All testing was done in the morning following a 12-hour fast and at least 24 hours after any caffeine ingestion. Subjects were instructed to maintain their training and not increase or decrease their volume or intensity over the course of the study. One hour prior to testing, cyclists ingested capsules containing either 6 mg of anhydrous caffeine per kilogram body weight or white flour (placebo) randomly administered in double-blind fashion. Time trials were performed on an indoor cycle trainer (Velotron; Racermate, Seattle, WA) on a computer-simulated course. The course consisted of eight laps of a flat, five-kilometer loop. Cyclists were free to self-select the resistance by changing gears during the test and were allowed to track distance completed on the course via a video display. However, they were blinded to their time, speed, and power output during the trials. Water was available for the cyclists to ingest ad libitum. Oxygen uptake and respiratory exchange ratio (RER) were obtained and averaged over the last two minutes of each lap. Heart rate and Ratings of Perceived Exertion (RPE; using the original 6-20 Borg scale) were obtained at the end of each lap.
Investigators were blinded to genotype until the subject completed the study. Furthermore, all genotyping was performed by an investigator not involved with the performance testing. DNA was obtained from whole blood samples via a QiaAmp mini-blood kit (Qiagen Inc.; Valencia, CA). Each blood sample was obtained prior to one of the cycling trials. Genotyping was performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), as previously described . Briefly, DNA was PCR amplified using the HotStar DNA Polymerase Kit (Qiagen) with the forward primer (5'-CAACCCTGCCAATCTCAAGCAC-3') and reverse primer (5'-AGAAGCTCTGTGGCCGAGAAGG-3') to generate a 920 bp fragment of the CYP1A2 gene. PCR conditions consisted of an initial denaturation at 95°C for 5 minutes, followed by 39 cycles at 94°C for 15 seconds, 64.5°C for 1 minute, and 72°C for 1 minute, with a final elongation step of 72°C for 10 minutes. One half of each PCR product was digested using the restriction enzyme ApaI (New England Biolabs, Ipswich, MA) as per manufacturer's instructions. Digested and undigested PCR products were evaluated in parallel via electrophoresis in a 2% agarose gel stained with ethidium bromide, and DNA bands were visualized by UV light. The presence of a 920 bp fragment following ApaI digestion identified the A/A genotype, while the presence of 709 bp and 211 bp fragments following ApaI digestion identified the C/C genotype. Caffeine metabolism is similar between heterozygotes and CC homozygotes . Therefore, similar to previous studies [11, 12], cyclists were grouped as AA homozygotes and C allele carriers; the latter group including both heterozygotes and CC homozygotes.
Descriptive data (height, weight, age, VO2max, caffeine intake) were compared between groups using independent t-tests. The frequency of low, moderate and high caffeine intake in the two genetic groups was compared using a Chi-Squared analysis. Potential differences in 40-km time, average VO2, HR, RER and RPE were assessed using repeated measures analysis of variance (RMANOVA) with treatment as a within-subjects factor and genotype as a between-subjects factor. For all RMANOVA procedures, post-hoc tests were performed using independent and dependent t-tests with a Bonferroni correction such that P < 0.025 was required for significance.