Precise determination of the effect of muscle temperature (T_m) on mitochondrial oxygen consumption kinetics has proven difficult in humans, in part due to the complexities in controlling for T_m-related variations in blood flow, fiber recruitment, muscle metabolism, and contractile properties. To address this issue, intracellular Po₂(P_iO2) was measured continuously by phosphorescence quenching following the onset of contractions in single Xenopus myofibers ( n = 24) while controlling extracellular temperature. Fibers were subjected to two identical contraction bouts, in random order, at 15°C (cold, C) and 20°C (normal, N; n = 12), or at N and 25°C (hot, H; n = 12). Contractile properties were determined for every contraction. The time delay of the P_iO2response was significantly greater in C (59 ± 35 s) compared with N (35 ± 26 s, P = 0.01) and H (27 ± 14 s, P = 0.01). The time constant for the decline in P_iO2was significantly greater in C (89 ± 34 s) compared with N (52 ± 15 s; P < 0.01) and H (37 ± 10 s; P < 0.01). There was a linear relationship between the rate constant for P_iO2kinetics and T_m( r = 0.322, P = 0.03). Estimated ATP turnover was significantly greater in H than in C ( P < 0.01), but this increased energy requirement alone with increased T_mcould not account for the differences observed in P_iO2kinetics among conditions. These results demonstrate that P_iO2kinetics in single contracting myofibers are dependent on T_m, likely caused by temperature-induced differences in metabolic demand and by temperature-dependent processes underlying mitochondrial activation at the start of muscle contractions.