Botulinum neurotoxin (BoNT) causes flaccid muscle paralysis at the neuromuscular junction (NMJ) by cleaving specific SNARE proteins in the presynaptic terminal. BoNT is well known as the agent responsible for causing botulism; however in the past two decades, the toxin has proven to be a valuable therapeutic tool in human medicine. BoNT injections have been used to successfully treat various neuromuscular disorders and currently, formulations of serotypes A and B are approved for specific uses. Although BoNT has proven to be an invaluable therapeutic agent, side effects of treatments include diffusion to undesired muscles, limited duration of treatment, and the development of resistance/tolerance with repeated exposure. In the current study, changes in SNARE protein chemistry following IM injections of BoNT/A (150kDa) were assessed to measure toxin diffusion and determine a temporal correlation with paralysis onset and recovery.