Exercise causes major shifts in multiple ions (e.g., K⁺, Na⁺, H⁺, lactate^-, Ca²⁺, and Cl^-) during muscle activity that contributes to development of muscle fatigue. Sarcolemmal processes can be impaired by the trans-sarcolemmal rundown of ion gradients for K⁺, Na⁺, and Ca²⁺ during fatiguing exercise, while changes in gradients for Cl^- and Cl^- conductance may exert either protective or detrimental effects on fatigue. Myocellular H⁺ accumulation may also contribute to fatigue development by lowering glycolytic rate and has been shown to act synergistically with inorganic phosphate (Pi) to compromise cross-bridge function. In addition, sarcoplasmic reticulum Ca²⁺ release function is severely affected by fatiguing exercise. Skeletal muscle has a multitude of ion transport systems that counter exercise-related ionic shifts of which the Na⁺/K⁺-ATPase is of major importance. Metabolic perturbations occurring during exercise can exacerbate trans-sarcolemmal ionic shifts, in particular for K⁺ and Cl^-, respectively via metabolic regulation of the ATP-sensitive K⁺ channel (K_ATP) and the chloride channel isoform 1 (ClC-1). Ion transport systems are highly adaptable to exercise training resulting in an enhanced ability to counter ionic disturbances to delay fatigue and improve exercise performance. In this article, we discuss (i) the ionic shifts occurring during exercise, (ii) the role of ion transport systems in skeletal muscle for ionic regulation, (iii) how ionic disturbances affect sarcolemmal processes and muscle fatigue, (iv) how metabolic perturbations exacerbate ionic shifts during exercise, and (v) how pharmacological manipulation and exercise training regulate ion transport systems to influence exercise performance in humans.