Exercise induces a post-exercise decline in the number of circulating lymphocytes. The aim of the present study was to investigate whether strenuous exercise induces lymphocyte apoptosis and generation of reactive oxygen species. Eleven healthy male subjects exercised for 2.5 h on a treadmill. Apoptotic lymphocytes were defined by being annexin positive and 7-aminoactinomycin-D negative. Measurement of F(2)-isoprostanes was used as a marker of oxidant stress in vivo. An increase (60%, P<0.05) in the percentage of apoptotic circulating lymphocytes was found 2 h post-exercise, whereas the total number of apoptotic cells did not change in relation to exercise. The concentration of plasma F(2)-isoprostanes increased approximately 1.6-fold in response to exercise, but declined towards pre-exercise values within the 1st h of recovery. The plasma concentrations of adrenaline, noradrenaline and cortisol increased during exercise. In conclusion, the results of the present study demonstrate that even in a study design in which high levels of apoptosis-inducing factors are generated, such as cortisol and isoprostanes, lymphocyte apoptosis does not contribute to post-exercise lymphocytopenia.