OBJECTIVE We investigated the acute effects of treadmill exercise test (TET) on total peroxide, total antioxidant capacity (TAC), oxidative stress index (OSI) and DNA damage levels in voluntary and untrained healthy subjects. METHODS A total of 113 untrained healthy subjects were included in the study. All subjects maintained a similar diet and physical activity for a week before the test. Blood samples were obtained before and after TET. Total peroxide, TAC, vitamin C and DNA damage were measured. The DNA damage was analyzed by using the Comet assay and OSI was calculated using total peroxide and TAC values. RESULTS Treadmill exercise test leads to the increase of total peroxide (12 +/- 3 micromol H2O2/L to 14 +/- 3 micromol H2O2/L, p<0.001), OSI (0.72 +/- 0.18 AU to 0.81 +/- 0.22 AU, p<0.001), and to the decrease of TAC (1.78 +/- 0.16 mmol Trolox Eq./L to 1.72 +/- 0.15 mmol Trolox Eq./L, p<0.001) and vitamin C levels (98 +/- 4.2 micromol/L to 95 +/- 3.4 micromol/L, p<0.001). There was not significant difference in DNA damage. CONCLUSIONS Our findings demonstrate that TET increases oxidants, decreases TAC and vitamin C namely, the balance shift towards oxidative side, but this stress is not enough to produce DNA damage.