Alterations in intracellular calcium (Ca2+) are thought to play an important role in skeletal muscle weakness associated with muscular dystrophy due to the activation of Ca2+-regulated proteases (calpains). It was hypothesized that impairments in Ca2+ regulation are exacerbated in dystrophic muscle and that calpain inhibition could attenuate the muscle weakness induced by fatiguing contractions. Single muscle fibres from control and dystrophic mice lacking dystrophin (mdx) and utrophin plus dystrophin (Utr-/-/mdx) were used. Fibres from Utr-/-/mdx mice had similar peak tetanic Ca2+ compared to control and mdx mice, however Utr-/-/mdx mice took longer to clear the released Ca2+. All fibres showed similar time to fatigue but fewer mdx and Utr-/-/mdx fibres remained excitable 1hr after fatiguing contractions. Exposure to a calpain inhibitor improved Ca2+ levels in dystrophic fibres (mdx; trend only in Utr-/-/mdx) after fatigue. Together, these data indicate that calpains play a role in prolonged muscle weakness after fatiguing contractions.