Living myocardial slices (LMSs) have shown great promise in cardiac research, allowing multicellular and complex interplay analyses with disease and patient specificity, yet their wider clinical use is limited by the large tissue sizes usually required. We therefore produced mini-LMSs (<10 mm²) from routine human cardiac surgery specimens and compared them with medium (10–30 mm²) and large (>30 mm²) slices. Size effects on biomechanical properties were examined with mathematical modeling, and viability, contraction profiles, and histological integrity were followed for 14 days. In total, 34 mini-, 25 medium, and 30 large LMS were maintained viable, the smallest measuring only 2 mm². Peak twitch force proved to be size-independent, whereas time-to-peak shortened as slice area decreased. Downsized LMSs displayed excellent contractile behavior for five to six days, after which a gradual functional decline and micro-architectural changes emerged. These findings confirm, for the first time, that mini-LMSs are feasible and viable, enabling short-term, patient-specific functional studies and pharmacological testing when tissue is scarce.