AIM: This study investigates the activation and regulation of phasic store-operated calcium entry (pSOCE) in fast- and slow-twitch skeletal muscle fibers. Specifically, we aimed to enhance the sensitivity of pSOCE detection in slow-twitch fibers by optimizing ionic conditions and to compare the physiological relevance of pSOCE between fiber types. METHODS: We employed mechanically skinned fast-twitch extensor digitorum longus (EDL) muscle fibers loaded with spectrally distinct Ca RESULTS: Skinned soleus fibers exhibited ~8-fold lower Ca CONCLUSION: These findings reveal a novel, fiber-type-specific difference in pSOCE regulation. When compared to EDL fibers, soleus fibers exhibited a higher sensitivity to SOCE activation despite releasing less Ca