Muscle fatigue is associated with a number of clinical diseases, including chronic pain conditions. Decreases in extracellular pH activates acid sensing ion channel 3 (ASIC3), depolarizes muscle, protects against fatigue, and produces pain. We examined if ASIC3-/- mice were more fatigable than ASIC3+/+ mice in a task-dependent manner. We developed two exercise protocols to measure exercise-induced muscle fatigue: (Fatigue Task 1:3, 1h runs; Fatigue Task 2: 3, 30min runs). In Fatigue Task 1, male ASIC3+/+ mice muscle showed less fatigue than male ASIC3-/- mice and female ASIC3+/+ mice. No differences in fatigue were observed in Fatigue Task 2. We then tested whether the development of muscle fatigue was dependent on sex and modulated by testosterone. Female ASIC3+/+ mice that were ovariectomized and administered testosterone developed less muscle fatigue than female ASIC3+/+ mice and behaved similarly to male ASIC3+/+ mice. However, testosterone was unable to rescue the muscle fatigue responses in ovariectomized ASIC3 -/- mice. Plasma levels of testosterone from male ASIC3-/- mice were significantly lower than in male ASIC3+/+ mice and were similar to female ASIC3+/+ mice. Muscle fiber types, measured by counting ATPase stained whole muscle sections, were similar in calf muscles from male and female ASIC3+/+ mice. These data suggest that both ASIC3 and testosterone are necessary to protect against muscle fatigue in a task-dependent manner. Also, differences in expression of ASIC3 and the development of exercise-induced fatigue could explain the female predominance in clinical syndromes of pain that include muscle fatigue.