Calorie restriction (CR) slows aging and consistently reduces circulating sex hormones in laboratory animals. However, nothing is known regarding the long-term effects of CR with adequate nutrition on serum sex-hormone concentration in lean healthy humans. In this study, we measured body composition, and serum total testosterone, total 17-β-estradiol, sex hormone–binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEA-S) concentrations in 24 men (mean age 51.5 ± 13 years), who had been practicing CR with adequate nutrition for an average of 7.4 ± 4.5 years, in 24 age- and body fat–matched endurance runners (EX), and 24 age-matched sedentary controls eating Western diets (WD). We found that both the CR and EX volunteers had significantly lower body fat than the WD volunteers (total body fat, 8.7 ± 4.2%; 10.5 ± 4.4%; 23.2 ± 6.1%, respectively; P = 0.0001). Serum total testosterone and the free androgen index were significantly lower, and SHBG was higher in the CR group than in the EX and WD groups (P ≤ 0.001). Serum 17β-estradiol and the estradiol:SHBG ratio were both significantly lower in the CR and EX groups than in the WD group (P ≤ 0.005). Serum DHEA-S concentrations were not different between the three groups. These findings demonstrate that, as in long-lived CR rodents, long-term severe CR reduces serum total and free testosterone and increases SHBG concentrations in humans, independently of adiposity. More studies are needed to understand the role of this CR-mediated reduction in sex hormones in modulating the pathogenesis of age-associated chronic diseases such as cancer and the aging process itself.