Increased levels of reactive oxygen and nitrogen species - as seen in response to exercise - challenges the cellular integrity. Important protective adaptive changes include induction of heat-shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, 21 young men were allocated into three groups receiving one of following oral supplementations: RRR--tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CE), RRR--tocopherol 290 IU/day + RRR--tocopherol 130 IU/day + AA 500 mg/day (CE), or placebo (CONTROL). After 28 days of supplementation, the subjects performed 3 hours of knee-extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), post exercise (3 h) and after a 3-hour recovery (6 h). In addition, blood was sampled for measurements of HSP72; -tocopherol; -tocopherol; AA; and 8-iso-prostaglandin-F₂ (8-PGF₂). Post supplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF₂, increased from 0 h to 3 h in all groups, however markedly less (P<0.05) in CE. In CONTROL, skeletal muscle HSP72 mRNA content increased 2.5-fold (P<0.05) and serum HSP72 protein increased 4-fold (P<0.05) in response to exercise, while a significant increase of skeletal muscle HSP72 protein content was not observed (P=0.07). In CE, skeletal muscle HSP72 mRNA, HSP72 protein and serum HSP72 were not different from CONTROL in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72 was completely blunted in CE. The results indicate that -tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.