Glycogen metabolism in exercising gastrocnemius muscles was examined by natural abundance 13C nuclear magnetic resonance (NMR) spectroscopy. Five-minute 13C-NMR measurement of muscle glycogen had a reproducibility of +/- 6.5% (+/- 4.8 mM). Experiments were performed on healthy fed male and female subjects. Two protocols were followed. 1) Subjects performed plantar flexion from rest at 15, 20, or 25% of maximum voluntary contraction for up to 9 h. 2) Subjects predepleted gastrocnemius glycogen with heavy exercise and then either performed low-intensity exercise as before or rested. Gastrocnemius glycogen was measured by NMR at rest and after each hour of exercise. In some sessions, both the exercised leg and the nonexercised leg were monitored with 13C-NMR. In protocol 1, blood velocity in the femoral artery was similarly assessed with ultrasonography. During low-intensity exercise from rest (protocol 1) muscle glycogen fell to a new steady-state value after several hours and then remained constant despite continued exercise. Mean blood velocity increased ninefold within 2 min of onset of exercise and remained constant thereafter. After predepletion (protocol 2), muscle glycogen was repleted both during low-intensity exercise and at rest. After 1 h the amount of glycogen repletion was greater when coupled with light exercise [48.5 +/- 2.8 mM after 1 h of exercise, 39.7 +/- 1.1 mM after 1 h of rest (P less than 0.05)]. During subsequent light exercise, glycogen reached a steady-state value similar to that obtained in protocol 1, while in resting, recovery glycogen levels continued to increase (+2.7 mM/h) over a 7-h period.(ABSTRACT TRUNCATED AT 250 WORDS)