Intense muscle contraction induces high rates of ATP hydrolysis with resulting increases in inorganic phosphate (P_i), H⁺ and ADP, factors thought to induce fatigue by interfering with steps in the cross-bridge cycle. Force inhibition is less at physiological temperatures; thus the role of low pH in fatigue has been questioned. Therefore, effects of pH 6.2 and collective effects with 30 mM P_i on the pCa-force relationship were assessed in skinned fast and slow rat skeletal muscle fibers at 15 and 30°C. At 30°C, the pH 6.2 and 30 mM P_i condition significantly depressed peak force in all fiber types with the greatest effect in type IIx fibers. Across fiber types, Ca²⁺ sensitivity was depressed by low pH and low pH plus high P_i, with the greater effect at 30°C. For type IIx fibers at 30°C, the pCa50 was 5.36 at pH 6.2 (no added P_i) and 4.98 at pH 6.2, 30 mM P_i, compared to 6.58 for control (pH 7, no added P_i). At 30°C, n2, reflective of thick filament cooperativity, was unchanged by low cell pH but was depressed from 5.02 to 2.46 in type IIx fibers with pH 6.2, 30 mM P_i. With acidosis, activation thresholds of all fiber types required higher free Ca²⁺ at both 15 and 30°C. With the exception of type IIx fibers, the Ca²⁺ required to reach activation threshold increased further with added P_i. In conclusion, it is clear that fatigue-inducing effects of low cell pH and elevated P_i at near-physiological temperatures are substantial.