It may be considered that the duration of intervals of high-intensity interval exercise (HIIE) can affect skeletal muscle biochemical and physiological responses. Also, sex-based differences in muscle biochemistry and physiology play a role in different responses to exercise. The aim of this study was to investigate the effects of sex differences on biochemical parameters involved in muscle glycolysis in response to HIIE protocols with short and long-term intervals. Forty male and female Wistar rats (aged 8 weeks, mean weighting 270 g and 225 g, respectively) were divided into HIIE with short-term interval (HIIESh), HIIE with long-term interval (HIIEL), and control groups. The phosphofructokinase (PFK), glycogen synthase 1 (GYS1), monocarboxylate transporter 4 (MCT4), and lactate dehydrogenase (LDH) levels were evaluated in gastrocnemius muscle after a single bout of exercise. The PFK, GYS1, and MCT4 levels were lower in HIIESh and HIIEL groups compared to the control group. However, there was no significant difference between sexes (P < 0.05). Moreover, the LDH activity was higher in both male and female HIIE rats compared to the control group. It is likely that HIIE with short- and long-term intervals represents similar effects on the glycolytic pathway in both male and female rats.