We discovered that treadmill exercise in-vivo promotes formation in skeletal fibers of new junctions between sarcoplasmic reticulum (SR) and transverse-tubules (TTs) containing STIM1 and Orai1, the two main players in store-operated Ca2+ entry (SOCE). We proposed that these new SR-TT junctions may function as Calcium Entry Units (CEUs) during repetitive muscle activity. Interestingly, in mice lacking Calsequestrin-1 (CASQ1-null) CEUs are constitutively present. In extensor digitorum longus (EDL) of mice, we quantified using electron-microscopy the number of CEUs/100μm2: 2.0±0.3 9.9±0.7; and 39.6±2.1, respectively in control (ctr)WT, exercised (ex)WT (subjected to 1 h of running at increasing speed: from 5 m/min to 25 m/min), and CASQ1-null mice. Higher number of CEUs/area in exWT and CASQ1-null mice correlates with higher expression levels of STIM1 and Orai1 detected by western-blot. We then used a repetitive stimulation protocol (30 x 1s-60Hz pulses every 5 seconds) to compare fatigue resistance in EDL muscles in presence or absence of extracellular Ca2+, or in presence of SOCE inhibitors (BTP-2, 2-APB and SKF-96365). Results of these experiments indicated: a) in 2.5 mM Ca2+ external solution, EDL muscles from exWT and CASQ1-null mice exhibited an increased capability to maintain contractile force compared to ctrWT mice (residual force after 15 tetani: 42.9±3.7%, 69.3±3.1% and 128.8±5.4% respectively for ctrWT, exWT, and CASQ1-null EDL muscles); b) in Ca2+-free external solution, muscles from exWT and CASQ1-null mice showed a greater decay of contractile force than ctrWT (residual force after 15 tetani: 30.4±2.4%, 34.7±3.3% and 63.5±2.2% respectively for ctrWT, exWT, and CASQ1-null EDL muscles); c) virtually identical results were obtained in presence of SOCE inhibitors. These data suggest that CEUs provide a preferential pathway for Ca2+ entry during repetitive muscle activity, likely important to limit muscle fatigue.