Circadian rhythms are known to influence muscular performance and regulate leptin hormone secretion, which assists in the regulation of body weight and energy homeostasis. In this study we examined the time-of-day effect on skeletal muscle eccentric contractile properties and the response of serum leptin to acute eccentric exhaustive isokinetic exercise. Male subjects (n = 26) performed a bout of exhaustive eccentric exercise on an isokinetic dynamometer in the morning or late afternoon time on different days. The test was repeated after 7-10 days at different time-of-day. Blood samples were collected at baseline and immediately after exercise for determination of serum leptin. Leg extensor muscle torque and work parameters were recorded, and compared with the time-of-day. Maximal eccentric torque values were not significantly affected by time-of-day. The maximal work performed during single repetition as well as total work values were significantly higher in the afternoon time. Leptin levels significantly decreased after exercise, but no time-of-day effect on serum leptin could be observed. Diurnal variations during short-term isokinetic exhaustive eccentric exercise seem to reflect muscle work capacity, with higher contractile efficiency towards the evening time. Although short-term exhaustive eccentric exercise decreased serum leptin, the levels did not to have a significant diurnal variation.