Advances in modeling and tracer techniques provide new perspective into glucose utilization and potential consequences to health or exercise performance. This study used stable isotope and compartmental modeling to evaluate how adaptation to a feed high in sugar and starch (SS) compared with a feed high in fat and fiber (FF) affects glucose kinetics at rest and during exercise in horses. Six trained Arabians adapted to each feed underwent similar tests at rest and while running approximately 4 m/s on a treadmill. For both tests, horses received 100 micromol/kg body weight [6,6-(2)H]glucose through a venous catheter. Circulating tracer glucose was described for 150 min by exponential decay curves and compartmental analysis. All parameters of glucose transfer increased with exercise (P < or = 0.004). Compared with FF horses, SS horses had higher circulating glucose (P = 0.022) and fractional glucose transfer rates (min(-1)) at rest (P = 0.055). Exercise increased glucose irreversible loss (mmol/min) more in SS horses (P = 0.037). Total glucose transfer during exercise tended to be greater in SS horses (0.027 +/- 0.002 mmol/min) compared with FF horses (0.023 +/- 0.002 mmol/min) (P = 0.109). This study characterized the effect of diet on glucose kinetics in resting and exercising horses using new modeling methods. Horses adapted to a fat-supplemented feed utilized less glucose during low-intensity exercise. Fat supplementation in horses may therefore promote greater flexibility in the selection of substrate to meet energy demands for optimal health and performance.