During the Gulf War, prophylactic treatment with pyridostigmine bromide (PB) along with the stress of deployment may have caused unexpected alterations in neural and immune function, resulting in a host of cognitive deficits which have become clinically termed Gulf War Illness (GWI). In order to test this interaction between PB and stress, the following study used a rodent model of GWI to examine how combinations of repeated restraint stress and PB induced alterations of peripheral cholinesterase (ChE) activity, corticosterone (CORT) levels, and cytokines on the last day of treatment, and then 10 days and three months post-treatment. Results indicate that PB decreases ChE activity acutely but sensitizes it by three months post-treatment selectively in rats subjected to stress. Similarly, while stress increased CORT levels acutely, rats in the PB/stressed condition continued to exhibit elevations in CORT at the delayed time point, indicating that PB and stress interact to progressively disrupt homeostasis in several peripheral measures. Because memory deficits are also common in clinical populations with GWI, we examined the effects of PB and stress on contextual fear conditioning. PB exacerbates stress-induced impairments in contextual fear conditioning ten days post-treatment, but protects against stress-induced augmentation of contextual fear conditioning at three months post-treatment. Collectively, these results provide critical insight as to how PB and stress may interact to contribute to the pathophysiological progression of GWI.