Abstract: Dynamic thiol–disulfide homeostasis is considered to have critical roles in maintenance of physiological functioning. We aimed to reveal whether there is any specific aberration in thiol–disulfide homeostasis in three distinct categories of individuals, including those who 1) exercise regularly (fitness group), 2) have a sedentary lifestyle (sedentary group) and 3) are overweight or obese (overweight/obese group). 72 male individuals were included in the study, 21 of whom were in fitness group, 28 of whom were overweight or obese and 23 of whom had a sedentary lifestyle. Plasma native thiol (-SH) and total thiol [(-SH) + (-S-S-)] levels were quantitatively determined. Total thiol levels in sedentary group were significantly lower than those in overweight/obese (p<0.05) and fitness groups (p<0.001). Also, disulfide values in fitness group were significantly higher than those in sedentary and overweight/obese groups (p<0.005, p<0.05). On the other hand, disulfide level, reduced and oxidized thiol ratios and oxidation/reduction ratio in fitness group differed significantly from the other groups (p<0.05). Thiol–disulfide homeostasis varies depending on lifestyle. The results of our study indicate that higher total thiol and disulfide levels are conspicuously distinctive features of thiol–disulfide homeostasis in individuals exercising regularly.