To evaluate the effects of exercise on net protein catabolism, the losses of urea in sweat and urine and urinary creatinine and 3-methylhistidine (3MH) excretion were determined in eight healthy subjects during cycle ergometer exercise performed at approximately 45% of VO2max for 90 min. The subjects ingested a meat-free diet for 5 days starting 3 days before and continuing for 1 day after the day of exercise. During exercise, total urea excretion (urine + sweat losses) increased 100% above pre- and postexercise values. Thirty percent of the total urea excretion during exercise was in the form of sweat losses. Total protein breakdown (as reflected by urea excretion), however, could account for less than 5% (21 +/- 4 kcal) of total calorie expenditure during the exercise (567 +/- 83 kcal). Urinary creatinine excretion increased by 50% during exercise. Urinary excretion of 3MH also tended to rise, but the ratio of urinary 3MH to creatinine showed no change in response to exercise. We conclude that 1) light to moderate exercise results in an increase in net protein catabolism and an increase in creatinine excretion; 2) sweat losses are an important route for urea excretion during exercise; 3) there is no evidence of a disproportionate increase in breakdown of myofibrillar contractile proteins; and 4) in spite of the increase in the rate of protein catabolism, protein is only a minor source of energy during light to moderate exercise.