Ten horses of Thoroughbred or Standardbred breeding were used to study the effects of dietary fish oil supplementation on the metabolic response to a high-intensity incremental exercise test. Horses were assigned to either a fish oil (n = 6) or corn oil (n = 4) treatment. The fish oil (Omega Protein, Hammond, LA) contained 10.6% eicosapentaenoic acid and 8% docosahexaenoic acid. Each horse received timothy hay and a textured concentrate at a rate necessary to meet its energy needs. The supplemental oil was top-dressed on the concentrate daily at a rate of 324 mg/kg BW. Horses received their assigned diet for 63 d, during which time they were exercised 5 d/wk in a round pen or on a treadmill. During wk 1, horses exercised for 10 min at a trot. After wk 1, exercise time and intensity were increased so that at wk 5, exercise time in the round pen increased to 30 min (10 min of cantering and 20 min of trotting) per day. Starting at wk 6, horses were exercised 3 d/wk in the round pen for 30 min and 2 d/wk on a treadmill for 20 min. After 63 d, all horses performed an exercise test consisting of a 5-min warm-up at 1.9 m/s, 0% grade, followed by a step test on a 10% grade at incremental speeds of 2 to 8 m/s. Blood samples were taken throughout exercise. During exercise, horses receiving fish oil had a lower heart rate (treatment x time interaction; P < 0.05) and tended to have lower packed cell volume (treatment effect; P = 0.087). Plasma lactate concentrations were not affected by treatment. Plasma glucose concentrations were not different between groups during exercise but were lower (treatment x time interaction; P < 0.01) for the fish oil group during recovery. Serum insulin tended to be lower in fish oil horses throughout exercise (treatment effect; P = 0.064). There was a tendency for glucose:insulin ratios to be higher for fish oil-treated horses throughout exercise (treatment effect; P = 0.065). Plasma FFA were lower (treatment x time interaction; P < 0.01) in horses receiving fish oil than in horses receiving corn oil during the initial stages of the exercise test. Serum glycerol concentrations also were lower in fish oil-treated horses (P < 0.05). Serum cholesterol concentrations were lower in horses receiving fish oil (treatment effect; P < 0.05), but serum triglycerides were not affected by treatment (P = 0.55). These data suggest that addition of fish oil to the diet alters exercise metabolism in conditioned horses.