While production of reactive oxygen and nitrogen species (RONS) is associated with some of the beneficial adaptations to regular physical exercise, it is not established whether RONS play a role in the improved insulin-stimulated glucose uptake in skeletal muscle obtained by endurance training. To assess the effect of antioxidant supplementation during endurance training on insulin-stimulated glucose uptake, twenty-one young healthy (age 29±1 y; BMI 25±3 Kg m(-2)) men were randomly assigned into either an antioxidant (AO; 500 mg vitamin C and 400 IU vitamin E (a-tocopherol) daily) or a placebo (PL) group that both underwent a supervised intense endurance-training program, 5 times per week for 12 weeks. A 3-hour euglycemic-hyperinsulinemic clamp, a maximal oxygen consumption (VO(2max)) and maximal power output (P(max)) test, and body composition measurements (fat mass, fat-free mass) were performed before and after the training. Muscle biopsies were obtained for determination of the concentration and activity of proteins regulating glucose metabolism. Although plasma levels of vitamin C (P <0.05) and a-tocopherol (P <0.05) increased markedly in the AO group, insulin-stimulated glucose uptake increased similarly in both the AO (17.2%, P <0.05) and the PL (18.9%, P <0.05) group in response to training. VO(2max) and P(max) also increased similarly in both groups (time effect: P <0.0001 for both) as well as protein content of GLUT4, hexokinase 2 and total Akt (time effect: P = 0.05 for all). Our results indicate that administration of antioxidants during strenuous endurance training has no effect on the training-induced increase in insulin sensitivity, in healthy individuals.