The purpose of this study was to evaluate the effect of elevated muscle vitamin E content on skeletal muscle damage from eccentric exercise. Sixty Sprague-Dawley rats were put on a normal (40 IU vitamin E/kg food) or supplemented (10,000 IU vitamin E/kg food) diet for 5 wk. Injury in soleus muscle was determined using several criteria: reductions in maximal tetanic force and number of intact fibers per square millimeter and elevations in muscle glucose 6-phosphate dehydrogenase activity and plasma creatine kinase activity, either immediately (0 h) or 2 days (48 h) after a downhill walking protocol. Sedentary animals were also tested but did not exercise. Muscle vitamin E levels were significantly elevated (approximately 3- to 4-fold), and susceptibility of the muscles to oxidant stress was decreased, after supplementation. However, vitamin E supplementation did not attenuate injury by any of the criteria employed. Maximal tetanic force decreased approximately 20% at 0 and 48 h after exercise in both groups. The number of intact fibers per square millimeter decreased approximately 30–35% in both groups at 0 and 48 h. Glucose 6-phosphate dehydrogenase activity increased approximately 50–100% in both groups at 48 h, and plasma creatine kinase activity was elevated approximately 2- to 2.5-fold at 0 h in both groups. These findings do not support a major role for free radical damage to muscle membranes in the initiation of injury from eccentric exercise, although they do not disprove free radical involvement in the etiology.