Antioxidant supplementation has been suggested to prevent exercise-induced muscle injury, but the findings are inconsistent. The objective of this study was to investigate the potential protective role of vitamin E treatment against eccentric exercise-induced muscle injury by examining morphological and functional alterations in rat soleus muscle after downhill running as well as muscle injury markers in the blood. Sixty adult male Wistar rats were randomly assigned to vitamin E-treated or placebo-treated groups studied at rest, immediately post-exercise or 48 h post-exercise (n = 10 per group). Vitamin E was administered by daily intraperitoneal injections of 100 mg/kg body mass of DL: -α-tocopheryl acetate for five consecutive days prior to exercise, resulting in the doubling of its plasma concentration. Downhill running resulted in significant (P < 0.05) changes in all injury markers for the placebo-treated rats at 0 and 48 h post-exercise. However, significantly smaller soleus muscle single-twitch tension (P (t)) and unfused (40 Hz) tetanic force, and greater plasma creatine kinase (CK) and lactate dehydrogenase (LD) activities compared with the control were found only immediately post-exercise for the vitamin E-treated rats (P < 0.05). Maximal tetanic force (P (o)) did not decline significantly compared to sedentary controls at neither time points measured. The vitamin E-treated rats had significantly (P < 0.05) higher soleus muscle P (t) immediately post-exercise than the placebo-treated rats as well as lower plasma CK and LD activity 48 h post-exercise. However, there was no difference in P (o) decline between groups at either time points measured. Vitamin E-treated rats had less pronounced morphological alterations in muscle in the immediate and 48-h post-exercise period. In conclusion, the effect of short-term vitamin E supplementation against eccentric exercise-induced muscle injury did not appear to be physiologically significant, because vitamin E failed to prevent the decline in the functional measure of P (o) compared to the placebo conditions.