Cuprizone induces massive demyelination in the CNS; however, the effects of cuprizone on the functions of the peripheral nervous system, as well as differential effects it may exert on males and females, have not yet been studied. Thus, we aimed to examine changes in the electromyographic characteristics of female and male Sprague-Dawley rats with cuprizone-induced demyelination. The body mass, food intake, nerve conduction velocity (NCV) along the sciatic nerve, latency, amplitude, and integral intensity (area) of the compound muscle action potential (CMAP; m. gastrocnemius) were measured weekly throughout a 4-week demyelination period, and histological sections of the brain and sciatic nerve were examined. Cuprizone led to a reduction in the NCV, CMAP amplitude and area, body mass, and food intake; simultaneously, it led to an increase in the CMAP latency. Notably, electrophysiological observations reveal that cuprizone affects both female and male rats, but males are more sensitive. This is the first study showing gender differences for the peripheral neurotoxicity of cuprizone.