Eight Thoroughbred horses were used to determine the effects of long-term calorie restriction and diet composition on serum T4 and T3 concentrations and metabolic responses with exercise. Horses were randomly assigned to 2 treatment groups (n = 4): Group 1, horses were fed a calorie-restricted diet designed to have 70% of the calories from the roughage source (RHR); Group 2, horses were fed a calorie-restricted diet designed to have 70% of the calories from the concentrate source (RHC). Horses then completed 2 step-wise exercise tests; one following a 12 h fast and one 2 h after a meal of 2 kg of a grain mix. Glucose concentrations declined (P < 0.01) in fed horses on the RHR diet but did not change in fed horses on the RHC diet. Fasted horses receiving the RHR diet had a more rapid increase in glucose concentration during exercise compared to fasted horses receiving the RHC diet (P < 0.01) as well as the highest glucose concentration at fatigue (P < 0.05). Insulin concentrations were higher (P < 0.05) at fatigue in fed horses on the RHR diet. Fasted horses receiving the RHR diet had higher (P < 0.01) pre-exercise FFA concentrations and a more rapid decline (P < 0.01) in FFA during exercise. Serum T3 concentrations increased (P < 0.01) in response to exercise within all treatments. The differences in thyroid hormone, glucose and FFA responses to exercise suggest that calorie source may be important in the hormonal regulation and energy metabolism of horses consuming calorie deficient diets.