The aim of this study was to evaluate in sedentary male subjects the effects of an acute bout of strenuous and moderate exercise on ex vivo platelet responsiveness and its possible relationship with exercise-associated modifications of oxidant-antioxidant status. An increased ADP- and collagen-evoked platelet aggregation associated with modified membrane fluidity and ion homeostasis was observed after exhaustive exercise. After moderate exercise, we found a decrease of platelet aggregation evoked by low concentrations of agonists. Strenuous exercise, but not moderate exertion, resulted in the enhanced accumulation of secondary products of lipid peroxidation, decreased total antioxidant capacity, including a diminished superoxide dismutase activity, and increased susceptibility of low-density lipoprotein (LDL) to in vitro oxidation. Acute elevation of plasma nitrite/nitrate (NOx) content was observed following each single session of physical test, whilst the platelet NOx content was decreased after strenuous exercise and increased after moderate exercise. Findings of the present study suggest that oxidative stress induced by acute strenuous exercise may interfere with platelet responsiveness most likely by promoting oxidized LDL-mediated platelet activation and by decreasing plasma and platelet-derived nitric oxide (NO) bioactivity. Moreover, our results further suggest that platelet responsiveness following an acute moderate physical stressor may depend on the efficiency of plasma and intraplatelet NO to desensitize platelets to agonist stimulation.