Exercise is known to increase reactive oxygen species, a condition recognized as oxidative stress. Obese individuals may experience even greater amounts of oxidative stress after exercise compared to normal weight people. It is not clear how weight loss affects exercise-induced oxidative stress in overweight subjects. The objectives of this study were to 1) evaluate the effect of the Army Physical Fitness Test (APFT) on biomarkers of oxidative stress in overweight/obese soldiers 2) determine the effects of dietary antioxidants, fitness level, body composition on exercise-induced oxidative stress, and 3) determine the effect of weight loss on changes in biomarkers of oxidative stress as a result of the APFT. A total of 60 subjects (35 M, 25 F) were recruited. After completing the 1st APFT (n=47), subjects followed a 3-month weight loss program and then completed the 2nd APFT (n=29). Blood measurements of the oxidative stress biomarkers creatine kinase (CK), C-reactive protein (CRP), glutathione peroxidase (GPX), and superoxide dismutase (SOD) were taken pre, immediately after and 24hrs after each exercise test. Dietary antioxidant intake, fitness level and body composition were also assessed at each APFT. After completing the 1st APFT, subjects showed a significant increase in CK and CRP levels immediately post-exercise and in CK at 24hrs post-exercise. There was a significant decrease in GPX immediately post-exercise but no significant change in SOD following exercise. Each of the oxidative stress biomarkers were found to be influenced by the antioxidant vitamins A, C and E, fitness level, total fat mass and total fat percentage. There were also significant interactions between fitness level and vitamins A, C, and E, and between fitness level and total fat mass and total fat percentage. There was no significant effect of attempted weight loss on the exercise-induced changes in the biomarkers, but there were significant changes in BMI, fat mass and fat percentage after the weight loss period. In conclusion, the APFT produced oxidative stress in overweight subjects which was not affected by attempted weight loss. Changes in oxidative stress biomarkers at the different time points were significantly affected by dietary antioxidants, fitness level, and body composition.