The endomysium, the innermost layer of the extracellular matrix (ECM), was shown to play an important role in both passive and active force transmission. However, its mechanical contribution within muscle fiber bundles remains incompletely understood. We hypothesized that dissecting one of the endomysial connections within a fiber bundle would alter force production and lead to changes in sarcomere lengths and their heterogeneity. Muscle fiber bundles (n = 12) were extracted from the extensor digitorum longus muscles of rats and chemically skinned. Their passive and active stress-length relationships were measured under two conditions:​ (i) with intact endomysium and (ii) after blunt dissection of endomysial connections between two adjacent peripheral fibers. Disruption of endomysial continuity significantly reduced passive stress by 26.5 % (p < 0.01) and active stress by 21.9 % (p < 0.001) on average across all lengths tested. It also shifted the optimal fiber length (L_opt) to longer values by 6.2 % (p < 0.01). Despite these mechanical changes, average sarcomere lengths (along fibers) and their heterogeneity remained unchanged. Length heterogeneity was only observed at a longer fiber length (1.3 L_opt) in the passive state before dissection. These findings demonstrate that endomysial connections are integral to the mechanical behavior of muscle fiber bundles, influencing both passive and active stress. The absence of sarcomere length changes along fibers suggests that other microstructural mechanisms, such as altered lattice spacing or impaired lateral force transmission, may underlie the observed changes. These results highlight the importance of ECM integrity in muscle mechanics and have implications for orthopedic interventions and neuromuscular disorder management.