Seven Arabian horses performed a standard incremental exercise test on a high-speed treadmill at 6% slope then were randomly assigned to two diets, a control diet of ground hay and concentrates and a similar diet with 10% added fat (by weight). Horses were sprint-trained 4 d/wk, and two additional exercise tests were performed at 5-wk intervals. Heart rates and rectal temperatures were monitored and venous blood samples were collected at rest and at each speed increment. Whole blood was analyzed for glucose, lactate, and hemoglobin concentrations, and plasma was analyzed for pH, pCO2, albumin, total protein, and sodium, potassium, and chloride concentrations. Bicarbonate concentration ([HCO3-]) and strong ion difference ([SID]) were calculated, and total weak acid ([Atot]) was estimated from total protein. During exercise, there were increases in plasma sodium and potassium concentrations (P < .001), whole blood lactate and glucose (P < .001), and hemoglobin concentrations (P < .01). There were decreases in plasma pH, [HCO3-], and chloride concentrations (P < .001). The decrease in plasma pH was associated with changes in [SID] and [Atot] that combined to offset a decrease in pCO2. After sprint training, heart rates at rest and during submaximal exercise were decreased (P < .01), whereas heart rates at the end of exercise were increased (P < .05). Sprint training also increased workrate and estimated oxygen consumption at a heart rate of 200 beats/min (P < .001). Training increased the duration of exercise and the speed attained at the end of exercise (P < .05). Training increased the blood hemoglobin response to exercise and decreased the pCO2 response (P < .01). There were diet x training interactions for pH, pCO2, and [SID] (P < .05). Horses consuming the high-fat diet had higher blood glucose during both standard exercise tests and higher lactate concentrations at fatigue (P < .05) during the last test. Fat adaptation involving sprint training of horses may influence glucolysis at the level of pyruvate during an incremental exercise test.