Feeding a high-fat diet increases fat utilisation and may decrease glycogen utilisation resulting in a lower lactate production during moderate exercise. The effects of fat feeding on exercise- and lipid metabolism-associated blood variables were evaluated in 6 Standardbred horses during submaximal exercise. The horses were fed a high- (11.8% fat in total dietary dry matter) or a low-fat diet (1.5% fat) in a cross-over experiment with feeding periods of 4 weeks. At the end of each feeding period, the horses were subjected to a submaximal standardised stepwise exercise test on a treadmill. Blood samples were obtained prior to the start, during the last 10 s of each exercise step, and after recovery (40 min walking in hand). Pre-exercise test values for plasma lactate, pH, pCO2, ionised sodium, ionised potassium, ionised calcium, ionised chloride, glucose, nonesterified fatty acids and glycerol did not differ for the 2 diets. The pre-exercise concentration of triacylglycerols was significantly lower on the high- compared to the low-fat diet (mean ± s.d. 0.17 ± 0.06 and 0.23 ± 0.08 mmol/l, respectively), whereas the concentrations of cholesterol (3.00 ± 0.47 and 2.11 ± 0.49 mmol/l, respectively), HDL cholesterol (1.80 ± 0.18 and 1.35 ± 0.27 mmol/l, respectively) and phospholipids (2.30 ± 0.11 and 1.72 ± 0.20 mmol/l, respectively) were significantly higher. There was significantly less plasma lactate accumulation during exercise when the horses were given the high-fat diet (P = 0.011). Horses on a low-fat diet had significantly different plasma lipid values when compared to being on a high-fat diet. The alterations in these values during and after moderate exercise were comparable for both diets. These results indicate that fat metabolism is significantly adapted on a high-fat diet; feeding such a diet may enhance potential performance by delaying lactate accumulation and thereby delaying the onset of fatigue.