This study investigated the changes in nitric oxide (NO) together with inducible nitric oxide synthase (iNOS) content and enzyme activity at 0, 4, 12, 24, and 48 h following acute muscle stretch injury. A single stretch injury was induced to the tibialis anterior muscle of 30 male New Zealand white rabbits (n = 6 at each time point). Injured and uninjured contralateral sham-operated muscles were harvested and analyzed for NO levels, iNOS content, and iNOS activity at each time point. Furthermore, three animals were used to estimate baseline NO levels and iNOS activity. There was a progressive reduction in NO content Nitric oxide (NO) generation occurs through the conversion of L-arginine to NO and L-citrulline, which is controlled by NO synthases (NOS) [1]. There are three forms of NOS that catalyze NO production [2]. NO is a ubiquitous molecule involved in a variety of physiological functions, including vascular regulation, neurotransmission, immune regulation, and cell signaling [3][4][5]. NO is also known to have profound effects on skeletal muscle function, including contractility, blood vessel tone, blood flow, and satellite cell activation [6][7][8][9]. Resting skeletal muscle contains two constitutive types, neural NOS (nNOS) and, to a lesser extent, endothelial NOS (eNOS) [7,10,11]. The third or inducible form (iNOS) is expressed mainly in leukocytes and macrophages in response to inflammation. In contrast to eNOS and nNOS, iNOS can produce large amounts of NO over a prolonged period [12,13],