OBJECTIVE n-3 fatty acids are known to exert multiple beneficial effects including anti-inflammatory actions that may diminish oxidative stress. Supplementation with antioxidant vitamins has been proposed to counteract oxidative stress and improve antioxidant status. Therefore, this project investigated the effects of daily supplementation in 48 trained cyclists over 6 wk and during 3 d of continuous exercise on F2-isoprostanes (oxidative stress), plasma n-3 fatty acids, and antioxidant status (oxygen radical absorption capacity and ferric-reducing antioxidant potential). METHODS Cyclists were randomized into n-3 fatty acids (N3) (n = 11) (2000 mg of eicosapentaenoic acid and 400 mg of docosahexaenoic acid), a vitamin-mineral (VM) complex (n = 12) emphasizing vitamins C (2000 mg), E (800 IU), A (3000 IU), and selenium (200 microg), a VM and n-3 fatty acid combination (VN3) (n = 13), or placebo (P) (n = 12). Blood was collected at baseline and preexercise and postexercise. A 4 x 3 repeated-measures ANOVA was performed to test main effects. RESULTS After exercise, F2-isoprostanes were higher in N3 (treatment effect P = 0.014). Eicosapentaenoic acid and docosahexaenoic acid plasma values were higher after supplementation (interaction effect P = 0.001 and 0.006, respectively) in both n-3 supplemented groups. Oxygen radical absorption capacity declined similarly among all groups after exercise. Ferric-reducing antioxidant potential exhibited significant interaction (P = 0.045) and significantly increased after exercise in VN3 and VM (P < 0.01). CONCLUSIONS This study indicates that supplementation with n-3 fatty acids alone significantly increases F2-isoprostanes after exhaustive exercise. Lastly, antioxidant supplementation augments plasma antioxidant status and modestly attenuates but does not prevent the significant n-3 fatty acid associated increase in F2-isoprostanes postexercise.