The repeated elevation of cytosolic Ca2+ concentration ([Ca2+]i) above resting levels during contractile activity has been associated with long-lasting muscle fatigue. The mechanism underlying this fatigue appears to involve elevated [Ca2+]i levels that induce disruption of the excitation-contraction (E-C) coupling process at the triad junction. Unclear, however, are which aspects of the activity-related [Ca2+]i changes are responsible for the deleterious effects, in particular whether they depend primarily on the peak [Ca2+]i reached locally at particular sites or on the temporal summation of the increased [Ca2+] in the cytoplasm as a whole. In this study, we used mechanically skinned fibers from rat extensor digitorum longus muscle, in which the normal E-C coupling process remains intact. The [Ca2+]i was raised either by applying a set elevated [Ca2+] throughout the fiber or by using action potential stimulation to induce the release of sarcoplasmic reticulum Ca2+ by the normal E-C coupling system with or without augmentation by caffeine or buffering with BAPTA. Herein we show that elevating [Ca2+]i in the physiological range of 2–20 μM irreversibly disrupts E-C coupling in a concentration-dependent manner but requires exposure for a relatively long time (1–3 min) to cause substantial uncoupling. The effectiveness of Ca2+ released via the endogenous system in disrupting E-C coupling indicates that the relatively high [Ca2+]i attained close to the release site at the triad junction is a more important factor than the increase in bulk [Ca2+]i. Our results suggest that during prolonged vigorous activity, the many repeated episodes of relatively high triadic [Ca2+] can disrupt E-C coupling and lead to long-lasting fatigue.