The aim of this study was to evaluate in sedentary individuals the effects of a 20-week exercise training program on ex vivo platelet responsiveness and the possible involvement of plasma antioxidant defences in relation to the mechanisms controlling platelet sensitivity. A statistically significant decrease in ADP- and collagen-evoked platelet aggregation was observed after physical training together with an increase in plasma total antioxidant capacity (TEAC), superoxide dismutase activity, and high-density lipoprotein cholesterol (HDL-C) concentration. Additionally, a rise in lag time for in vitro low-density lipoprotein (LDL) oxidation as well as a decreased plasma level of secondary products of lipid peroxidation were observed after training, and the values for lag time were significantly correlated with TEAC and HDL-C. Nitrate/nitrite (NOx) content both in plasma and in platelet cytosol was significantly enhanced at the end of the training period and a significant positive correlation was found between plasma and intraplatelet NOx values. Furthermore, intraplatelet NOx content was positively correlated with HDL-C levels. The findings of the current study suggest that the improvement of antioxidant defences induced by moderate regular exercise may be involved in desensitising blood platelets most likely through the inhibition of LDL oxidation and the simultaneous enhancement of plasma and intraplatelet NOx bioavailability and HDL-C level.