Tenderness is a key determinant of consumer acceptance of beef. Electrical stimulation (ES) is commonly used to improve tenderness by reducing the impact of cold-shortening but also disrupts muscle structure, accelerates pH decline, and augments calcium release, which may influence calpain-1 mediated proteolysis. Evidence supporting increased proteolysis with ES is inconsistent. To investigate the relationship, we examined the effects of ES on the calpain-1 proteolytic system. Market-weight steers were harvested, carcasses were split, and one side was subjected to ES (15 Hz and at 150 or 300 V) and the contralateral side served as a non-stimulated (NS) control. Samples from longissimus thoracis et lumborum (LTL) and semitendinosus (ST) were collected pre- and post-ES, and at 1, 3, 6, 9, 24 h, and 3, 5, 7, 14, and 21 d postmortem. ES accelerated pH decline (P < 0.001) and calpain-1 autolysis, showing the most rapid loss (P < 0.001) of the 80 kDa subunit. In the LTL, ES led to a greater (P < 0.014) abundance of the calpain-1 76 kDa subunit, while the ST had a faster and greater accumulation (P < 0.006) of the calpain-1 76 kDa subunit. Additionally, ES increased (P < 0.031) the rate of calpastatin degradation in the ST. Interestingly, ES carcasses did not display greater degradation of desmin or troponin-T over aging compared to controls. These results indicate that although ES accelerates pH decline and promotes calpain-1 activation and calpastatin degradation, it does not enhance proteolysis. Thus, improvements in tenderness following ES are likely driven by structural disruption rather than increased proteolysis.