The effect of endurance exercise on enhancing insulin sensitivity and glucose flux has been well established with techniques such as the hyperinsulinemic clamp. Although informative, such techniques do not emulate the physiological postprandial state, and it remains unclear how exercise improves postprandial glycaemia. Accordingly, combining mixed meal tolerance testing and the triple-stable isotope glucose tracer approach, glucose fluxes (rates of meal glucose appearance (Ra), disposal (Rd) and endogenous glucose production (EGP)) were determined following acute endurance exercise (1h cycling; ~70% VO2max) and 4 weeks of endurance training (cycling 5d/wk). Training was associated with a modest increase in VO2max (~7%, P<0.001). Postprandial glucose and insulin responses were reduced to the same extent following acute and chronic training. Interestingly, this was not accompanied by changes to rates of meal Ra, Rd or degree of EGP suppression. Glucose clearance (Rd relative to prevailing glucose) was, however, enhanced with acute and chronic exercise. Furthermore, the duration of EGP suppression was shorter with acute and chronic exercise, with EGP returning toward fasting levels more rapidly than pre-training conditions. These findings suggest that endurance exercise influences the efficiency of the glucoregulatory system, where pre-training rates of glucose disposal and production were achieved at lower glucose and insulin levels. Notably, there was no influence of chronic training over and above that of a single exercise bout, providing further evidence that glucoregulatory benefits of endurance exercise are largely attributed to the residual effects of the last exercise bout.