The purpose of this study was to evaluate the hypotheses that accumulation of hydrogen ions and/or inorganic phosphate (Pi) in skeletal muscle increases with repeated bouts of isotonic exercise. ³¹P-Magnetic resonance spectroscopy was used to examine the gastrocnemius muscle of seven highly aerobically trained females during four bouts of isotonic plantar flexion. The exercise bouts (EX1-4) of 3 min and 18 s were separated by 3 min and 54 s of complete rest. Muscle ATP did not change during the four bouts. Phosphocreatine (PCr) degradation during EX1 (13.3 ± 2.4 mmol/kg wet weight) was higher (P < 0.01) compared with EX3-4 (9.7 ± 1.6 and 9.6 ± 1.8 mmol/kg wet weight, respectively). The intramyocellular pH at the end of EX1 (6.87 ± 0.05) was significantly lower (P < 0.001) than those of EX2 (6.97 ± 0.02), EX3 (7.02 ± 0.01), and EX4 (7.02 ± 0.02). Total Pi and diprotonated Pi were significantly higher (P < 0.001) at the end of EX1 (17.3 ± 2.7 and 7.8 ± 1.6 mmol/kg wet weight, respectively) compared with the values at the end of EX3 and EX4. The monoprotonated Pi at the end of EX1 (9.5 ± 1.2 mmol/kg wet weight) was also significantly higher (P < 0.001) than that after EX4 (7.5 ± 1.1 mmol/kg wet weight). Subjects' rating of perceived exertion increased (P < 0.001) toward exhaustion as the number of exercises progressed (7.1 ± 0.4, EX1; 8.0 ± 0.3, EX2; 8.5 ± 0.3, EX3; and 9.0 ± 0.4, EX4; scale from 0 to 10). The present results indicate that human muscle fatigue during repeated intense isotonic exercise is not due to progressive depletion of high energy phosphates nor to intracellular accumulation of hydrogen ions, total, mono-, or diprotonated Pi.