The development of a sensitive radioimmunoassay for the measurement of myoglobin now permits the accurate determination and detection of even small concentrations of serum myoglobin. To determine the effect of exercise and physical conditioning on serum myoglobin levels, 16 male Army recruits (ages 18 to 28 years) underwent treadmill exercise and strenuous physical evaluation tests (PETs) that consisted of sit-ups, push-ups, horizontal ladder, obstacle course, and 3.2-km run. These tests and exercise were performed before and after 12 weeks of physical conditioning performed in conjunction with basic military training. Serum myoglobin levels did not rise after treadmill exercise, but they were increased greatly six hours after PET. After physical conditioning, serum myoglobin concentrations measured after the treadmill exercise remained normal and values after PET showed a significant decrease (P less than 0.001) as compared with those measured before physical conditioning (mean +/- SD, 160 +/- 81 ng/mL after initial PET vs 76 +/- 60 ng/mL after physical conditioning). These results demonstrate that physical training reduces the degree of myoglobinemia occurring after strenuous exercise, possibly by modifying skeletal muscle alterations that allow myoglobin release in this setting.