Testosterone exerts a widespread pattern of effects on metabolism and body composition, and interest is gaining in its correlation with physical fitness. The main focus of our study was to investigate the association of total serum testosterone and sex hormone-binding globulin (SHBG) levels on exercise capacity and maximal power output in men using a cross-sectional, population-based adult cohort. From the Study of Health in Pomerania (SHIP), 624 men age 25 to 85 years who underwent a standardized progressive incremental exercise protocol on a cycle ergometer were included in the analyses. Exercise capacity was characterized by oxygen uptake at anaerobic threshold (V'O(2) at L) and peak exercise (V'O(2 peak)) as well as maximal power output at peak exertion. Multivariable linear regression analyses adjusted for age, sex, body mass index, physical activity, and smoking were performed. Further, linear regression analyses with cubic splines and sensitivity analyses were undertaken. At peak exercise performance, testosterone and SHBG levels showed no associations with V'O(2 peak), V'O(2) at L as well as maximal power output, even after controlling for confounding factors including age, body mass index, physical activity, and smoking. An adverse association between the free testosterone index and V'O(2) at L was found. Linear regression analyses with cubic splines did not change the main results. In conclusion, this is the first study focusing on the association of total serum testosterone and SHBG on exercise capacity and physical performance in healthy volunteers based on a large-scale population-based study. After adjustment for relevant influencing factors, neither total serum testosterone nor SHBG levels had any interference with peak exercise capacity, aerobic exercise capacity, or maximal power output in men.