OBJECTIVE To determine glycogen resynthesis rate and changes in plasma metabolite concentrations in horses before and after repeated exercise. METHODS 6 clinically normal Standardbred trotters. METHODS Horses trotted distances of 3,000, 3,000, and 2,000 m (trial A) and 3 days later, trotted 2,100, 2,100, and 1,600 m (trial B). Horses had 1 hour rest periods between bouts of exercise. Trotting speed was increased with each exercise bout, up to a near maximal. Muscle biopsy specimens and venous blood samples were obtained before each trial and 0, 4, 24, 48, and 72 hours after the third bout. Blood samples were also taken between exercise bouts. Muscle glycogen content and plasma glucose, glycerol, nonesterified fatty acid, and triglyceride concentrations were determined. RESULTS Muscle glycogen content was significantly decreased immediately after exercise from 473 +/- 45 to 329 +/- 79 mmol/kg of dry weight in trial A, and from 472 +/- 128 to 347 +/- 59 mmol/kg in trial B. Further decreases were measured 4 hours after exercise. Glycogen resynthesis was negligible 24 hours after exercise. Basal muscle concentrations of glycogen were obtained 72 hours after exercise in trial A (472 +/- 128 mmol/kg), but not in trial B (279 +/- 52 mmol/kg). Plasma concentrations of glucose were greater than or equal to before-exercise values. Plasma concentrations of lipid metabolites, glycerol, triglycerides, and nonesterified fatty acids, were less than before-exercise values 2 to 72 hours after exercising. CONCLUSIONS Repeated bouts of exercise decrease glycogen repletion rate, which is not attributable to hypoglycemia, but may be influenced by limited availability of lipids for energy production.