The effect of electrolyte supplementation on plasma electrolyte profile in horses in hot environmental conditions was studied using sweat loss and sweat electrolyte concentration. Eleven adult Marwari mares were selected for the study. To develop an electrolyte supplement, 7 mares were given a preliminary 20 km moderate intensity exercise and the electrolyte supplement was designed on basis of their body weight loss and sweat electrolyte concentration during exercise. In the subsequent trials, 4 mares were kept in an exercise control group that did not receive the supplement (ECG), while 3 mares were kept in the exercise supplementation group (ESG) that received the supplement. Four other mares were used as resting controls (RG). ESG mares were daily fed 50 g electrolyte supplement and 150 g supplement orally 1 h before the 20 km trial. Mares of ESG and ECG groups were conferred 3 km trot + canter riding every day in the morning and a 20 km trot + canter ride on every 10th day of the total trial period of 40 days. Blood analysis revealed a significant (P<0.05) decline in plasma calcium and chloride concentrations immediately after exercise in both groups. There was significant less post-exercise decrease of plasma calcium and chloride concentration in ESG mares. Post-exercise plasma sodium concentration was higher (P<0.05) and plasma potassium concentration was lower only in ESG mares (P<0.05) compared to pre-exercise concentrations. The supplement did not affect the physiological responses (heart rate and rectal temperature). However, the changes in plasma sodium, potassium, calcium and chloride concentration were in favour of better performance in supplemented (ESG) mares and advocate sweat-developed electrolyte supplementation in riding mares.