The purpose of the present study was to determine whether exposure to exogenous physiological concentrations of caffeine influence contractility, Ca²⁺ handling, and fatigue development in isolated single Xenopus laevis skeletal muscle fibers. After isolation, two identical contractile periods (separated by 60 min rest) were conducted in each single myofiber (n=8) at 20° C. During the first contractile period, four fibers were perfused with a non-caffeinated Ringer's solution, while the other four fibers were perfused with a caffeinated (70 µM) Ringer's solution. The order was reversed for the second contractile period. The single myofibers were stimulated during each contractile period at increasing frequencies (0.16, 0.20, 0.25, 0.33, 0.50, and 1.0 tetanic contractions/second) with each stimulation frequency lasting 2 minutes until fatigue ensued, defined in this study as a fall in tension development to 66% of maximum. Tension development and free cytosolic [Ca²⁺] (fura-2 fluorescence spectroscopy) were simultaneously measured. There was no significant difference in the peak force generation, time to fatigue, cytosolic Ca²⁺ levels, or relaxation times between the non-caffeinated and caffeinated trials. These results demonstrate that physiological levels of caffeine have no significant effect on Xenopus single myofiber contractility, Ca²⁺ handling, and fatigue development and suggest that that any ergogenic effects of physiological levels of caffeine on muscle performance during contractions of moderate-to-high intensity are likely related to factors extraneous to the muscle fiber.