The time course of insulin sensitivity, skeletal muscle glycogen and GLUT4 contents, and glycogen synthase activity (GS) after a single bout of intense exercise was examined in 8 horses. A euglycemic hyperinsulinemic clamp (EHC) was undertaken at one of 0.5, 4 or 24 h after exercise or after 48 h of rest (Con) and mean glucose infusion rate (GIR) was determined. There was no increase in GIR with exercise but GIR was significantly decreased at 0.5 h (8.6 ± 2.7, 6.7 ± 2.0, 9.0 ± 2.0, and 10.6 ± 2.2 mg/kg/min for Con, 0.5, 4, and 24 h, respectively). Muscle glycogen content (pre-EHC; mmol glucosyl units/kg dm) was higher (P<0.05) in Con (565 ± 102) than in other treatments with values of 317 ± 84, 362 ± 79 and 382 ± 74 for 0.5, 4, and 24 h, respectively. Before the EHC, active:total GS activity ratio was higher (P<0.05) at 0.5, 4 and 24 h than in Con. Active GS and active:total GS activity ratio were higher in (P<0.05) in Con and 24 h post EHC. There was a significant inverse correlation (r = -0.43, P=0.02) between glycogen content and GS activity ratio, but no relationship between GS activity and GIR. Total muscle GLUT4 content was unchanged following exercise. Insulin sensitivity was not increased by prior exercise that resulted in a significant reduction in muscle glycogen content in horses. This lack of increase in insulin sensitivity is consistent with the slow rate of muscle glycogen resynthesis observed in equine studies.