Muscles produce oxidants, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), from a variety of intracellular sources. Oxidants are detectable in muscle at low levels during rest and at higher levels during contractions. RNS depress force production but do not appear to cause fatigue of healthy muscle. In contrast, muscle-derived ROS contribute to fatigue since loss of function can be delayed by ROS-specific antioxidants. Thiol regulation appears to be important in this biology. Fatigue causes oxidation of glutathione, a thiol antioxidant in muscle fibers, and is reversed by thiol-specific reducing agents. N-acetylcysteine (NAC), a drug that supports glutathione synthesis, has been shown to lessen oxidation of cellular constituents and delay muscle fatigue. In humans, NAC pretreatment improves performance of limb and respiratory muscles during fatigue protocols and extends time-to-task failure during volitional exercise. These findings highlight the importance of ROS and thiol chemistry in fatigue, show the feasibility of thiol-based countermeasures, and identify new directions for mechanistic and translational research.