Leukocyte telomeres shorten with age, and excessive shortening is associated with age-related cardiometabolic diseases. Exercise training may prevent disease through telomere length maintenance although the optimal amount of exercise that attenuates telomere attrition is unknown. Furthermore, the underlying molecular mechanisms responsible for the enhanced telomere maintenance observed in endurance athletes is poorly understood. We quantified the leukocyte telomere length and analyzed the expression of telomere-regulating genes in endurance athletes and healthy controls (both n = 61), using quantitative PCR. We found endurance athletes have significantly longer (7.1%, 208-416 nt) leukocyte telomeres and upregulated TERT (2.0-fold) and TPP1 (1.3-fold) mRNA expression compared with controls in age-adjusted analysis. The telomere length and telomere-regulating gene expression differences were no longer statistically significant after adjustment for resting heart rate and relative V̇o2 max (all P > 0.05). Resting heart rate emerged as an independent predictor of leukocyte telomere length and TERT and TPP1 mRNA expression in stepwise regression models. To gauge whether volume of exercise was associated with leukocyte telomere length, we divided subjects into running and cycling tertiles (distance covered per week) and found individuals in the middle and highest tertiles had longer telomeres than individuals in the lowest tertile. These data emphasize the importance of cardiorespiratory fitness and exercise training in the prevention of biological aging. They also support the concept that moderate amounts of exercise training protects against biological aging, while higher amounts may not elicit additional benefits.