Heart rate and Ratings of Perceived Exertion (RPE; using the original 6-20 Borg scale) were obtained at the end of each lap. Genotyping Investigators were blinded to genotype until the subject completed the study. Furthermore, all genotyping was performed by an NVP-BSK805 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

[12]. 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 mTOR inhibitor 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 [10]. 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. Pyruvate dehydrogenase Statistical analyses 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. Results Out of the 35 participants analyzed, 16 (46%) were homozygous for the A variant and 19 (54%) were C allele Angiogenesis inhibitor carriers. This distribution is very similar to previously reported studies [10–12, 15]. Descriptive characteristics of the two genotype groups are shown in Table 1. There were no significant differences (p > 0.05) between the two groups for height, weight, age, VO2max, or caffeine intake.