We studied 31P-nuclear magnetic resonance spectroscopy (31P-MRS) for repeated non-invasive measurement of changes in the muscle metabolites, i.e., creatine phosphate (PCr) and inorganic phosphate (Pi), and changes in intramuscular pH, during repeated exercise and intervening rest periods. Six healthy male subjects performed 2 min of femoral flexion exercise at 20 kgm/min in a 2.1 Tesla superconducting magnet with a 67 cm bore. This exercise was repeated 4 times with 2-min rest periods between bouts. During this time 31P-MRS data was collected with 32 scans per spectrum, requiring 12.8 sec. During exercise, the PCr decreased to 30.3 +/- 7.0% (mean +/- SD) of the pre-exercise level, and it did not completely recover during the rest period (79.0 +/- 1.3% of the pre-exercise level). Thereafter, when the exercise was repeated, the lowest PCr value during exercise decreased gradually (2nd bout, 22.1 +/- 3.9%; 3rd bout, 16.7 +/- 3.2%, and 4th bout 14.9 +/- 3.5%). The Pi increased by 705-740% during exercise and there were no significant differences in maximum Pi among the 4 bouts. During the rest period after the first bout, Pi fell to about 10% below the pre-exercise level in four of the six subjects. With repeated exercise, this undershoot was less common. The Pi/PCr ratio during exercise increased linearly over time. Furthermore, the maximum Pi/PCr ratio during exercise increased significantly with repeated exercises. We conclude that as exercise is repeated PCr gradually decreases, but there is no cumulative increase in Pi. Thus, the Pi/PCr ratio, an indicator of free ADP, increased with repeated exercise.