The objectives of this study were to compare sweat collection techniques and determine effect of low and high intensity exercise on the composition and osmolality of equine sweat in exercise trained Thoroughbred horses. Sweat collection sites included the neck and thorax, bilaterally and behind the girth ventrally. Sweat was collected in cotton gauze, either exposed to the air or contained within a plastic pouch, from sealed pouches, with or without application of vaseline to the skin, and as it dripped off the skin behind the girth (free catch). There were differences in the ionic composition of sweat collected by different techniques during and after low (50% V̇O2max) and after high (90% V̇O2max) intensity exercise. These changes were most consistent where collection technique minimised evaporative losses. With regard to collection method, ionic concentrations and osmolality were lowest in samples obtained from sealed pouches without vaseline on the skin. In samples obtained from cotton gauze and by free catch, osmolality, [Na] and [Cl] were significantly increased when compared with sweat collected from sealed pouches. In samples collected from sealed pouches during low intensity exercise, osmolality increased 11%, [Na] and [Cl] increased 26% and 9%, respectively and [K] decreased 14%. Osmolality and the concentrations of these ions did not change during recovery. In contrast, osmolality increased by 12% in the first 5 min following high intensity exercise. After both intensities of exercise, there was a significant decrease in the concentration of protein in sweat collected by all methods. Results indicate that collection technique had a marked effect on composition of sweat as demonstrated by increased osmolality and increased concentrations of Na and Cl in sweat collected in cotton gauze and by free catch.