Background Negative-pressure wound therapy (NPWT) can improve fasciotomy wound closure, but its effects on skeletal muscle are largely unknown. The purpose of this study was to evaluate NPWT effects on skeletal muscle after fasciotomy for compartment syndrome in an animal model and to assess regional variability in muscle fiber regeneration. Methods Compartment syndrome was induced in the hindlimb of twenty-two adult female pigs with use of a continuous intracompartmental serum-infusion model. Fasciotomy was performed after six hours, and animals were randomized to receive either wet-to-dry gauze dressings (control group) or NPWT dressings (−125mm Hg, continuous suction) for seven days. Delayed primary wound closure was attempted at seven days, and the peroneus tertius was harvested for analysis seven days or twenty-one days after fasciotomy. Muscles were weighed, and hematoxylin and eosin-stained samples from four regions of the muscle (superficial central, deep central, lateral, and proximal) were mapped for different cellular morphologies. Results Muscle weight was greater in the affected limb at all time points with no difference between treatment groups. At seven days, only the deep central samples in the NPWT group had a significantly greater cross-sectional area containing normal fibers as compared with that found in the controls. By twenty-one days, the deep central, lateral, and proximal regions of the NPWT-treated muscles had a smaller cross-sectional area containing normal fiber morphology and a greater cross-sectional area containing only mononucleated cells as compared with the controls. Conclusions NPWT did not decrease muscle weight. At twenty-one days, the extent of muscle fiber regeneration after fasciotomy for compartment syndrome was reduced in muscles treated with NPWT for seven days compared with the values in the control group treated with wet-to-dry gauze dressings. Clinical Relevance NPWT may be harmful to skeletal muscle after compartment syndrome requiring fasciotomy and local wound care.