The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the decrease in pH during an identical, subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers (n=9) were isolated from Xenopus lumbrical muscle, and incubated with the fluorescent cytosolic H⁺ indicator BCECF AM for 30 min. Two identical contractile periods were performed in each fiber, separated by a one-hour recovery period. Force and intracellular pH (pH_i) fluorescence were measured simultaneously while fibers were stimulated (tetanic contractions of 350 ms trains with 70 Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5 and 1 contraction/sec) until the development of fatigue (to 60% initial force). No significant difference (p<0.05) was observed between the first and second contractile periods in initial force development, resting pH_i, or in the time to fatigue (5.3±0.5 vs 5.1±0.6 min). However, the relative decrease in the BCECF fluorescence ratio (and therefore pH_i) from rest to the fatigue time point was significantly greater (P<0.05) during the first contractile period (to 65±4% of initial resting values) when compared to the second (77±4%). The results of the present study demonstrated that, when preceded by an initial fatiguing contractile period, the rise in cytosolic [H⁺] in contracting single skeletal muscle fibers during a second contractile period was significantly reduced, but did not attenuate the fatigue process in the second contractile period. These results suggest that intracellular factors other than H⁺ accumulation contribute to the fall in force development under these conditions.