This study examined the effect of alterations in rat intramuscular connective tissue (CT), secondary to limb immobilization, on the muscle's susceptibility to contraction-induced injury. Hindlimbs were casted for 3 wk with the extensor digitorum longus muscle fixed in a shortened (IM-SP) or lengthened position (IM-LP). An age-matched control group remained uncasted. Extensor digitorum longus muscles were injured in vivo by using a motorized foot pedal that repeatedly flexed and extended the foot while the muscle was electrically stimulated during plantar flexion. Four hours postinjury, maximum isometric tetanic force (Po) was measured in vitro and was used as a functional index of muscle injury. Muscles were fixed, sectioned, and stained for later analysis. Intramuscular CT concentration, expressed as the ratio of CT area to muscle fiber area, was significantly higher in both IM-SP (0.153 +/- 0.003) and IM-LP (0.174 +/- 0.003) groups compared with controls (0.104 +/- 0.003). Po values of injured muscles both IM-LP and IM-SP were higher than the injured controls' Po of 9.41 +/- 0.63 N/cm2 (P < 0.05). Injured IM-LP muscle forces were significantly higher than those of IM-SP. This study demonstrated that limb immobilization increases intramuscular CT concentration, which is accompanied by attenuation of muscle injury. We conclude that remodeling of intramuscular CT affects the muscle's resistance to contraction-induced injury.