DescriptionImpaired hepatic triglyceride (TG) metabolism is associated with dysfunctions such as insulin resistance and elevated VLDL-TG secretion. Chronic exercise lowers plasma TG and hepatic TG, however, many benefits of chronic exercise are due to repeated effects of single exercise sessions. Little is known, however, about effects of acute exercise on hepatic TG metabolism or the influence of exercise intensity in males vs. females. We examined impacts of single bouts of moderate-intensity continuous exercise (CE) vs. high-intensity interval exercise (HIIE) on hepatic TG metabolism and secretion in mice of both sexes. Hepatic TG was transiently increased on the day of exercise and in females the increase was greater with HIIE. These exercise-related changes appeared driven by enhanced transcription of the lipid-droplet coating protein Perilipin 2. On the day after exercise, VLDL-TG secretion rate was only reduced by HIIE in females. These findings demonstrated intensity-dependent and sex-specific effects of acute exercise. Because of our findings that single bouts of HIIE alter hepatic TG metabolism more than CE and the potential for chronic exercise to attenuate severity of complications associated with menopause, we compared 6 weeks of training using these exercise types on hepatic TG metabolism and secretion, glucose tolerance, body composition, and strength in ovariectomized (OVX) and sham-operated (SHAM) mice. Additionally, we measured energy expenditure and substrate oxidation during and immediately after exercise and assessed post-exercise spontaneous physical activity (SPA) to further characterize these exercise modalities. In OVX and SHAM, CE and HIIE elicited similar energy expenditures during exercise and in the post-exercise period lowered absolute carbohydrate oxidation and SPA. OVX vs. SHAM displayed impaired glucose tolerance, elevated blood glucose, and greater body fat despite lower hepatic TG, as well as lower strength, and these outcomes were not affected by training. In contrast to responses to single exercise sessions, chronic HIIE increased hepatic AMPK protein. Further, the reduction in VLDL-TG secretion after a single HIIE session was not maintained after training. These results reveal intensity-dependent effects of habitual exercise on hepatic AMPK expression and with our findings of single bouts reveal distinct responses in hepatic TG metabolism to acute vs. chronic exercise.