Carbohydrate stores within muscle are considered essential as a fuel for prolonged endurance exercise, and regimes for enhancing such stores have proved successful in aiding performance. This study explored the effects of a hyperglycaemic-hyperinsulinemic clamp performed 18 h previously on subsequent prolonged endurance performance in cycling. Seven male subjects, accustomed to prolonged endurance cycling, performed 90 min of cycling at ~65% VO(2max) followed by a 16-km time trial 18 h after a 2-h hyperglycemic-hyperinsulinemic clamp (HCC). Hyperglycemia (10 mM) with insulin infused at 300 mU/m(2)/min over a 2-h period resulted in a total glucose uptake of 275 g (assessed by the area under the curve) of which glucose storage accounted for about 73% (i.e. 198 g). Patterns of substrate oxidation during 90-min exercise at 65% VO(2max) were not altered by HCC. Blood glucose and plasma insulin concentrations were higher during exercise after HCC compared with control (p < 0.05) while plasma NEFA was similar. Exercise performance was improved by 49 s and power output was 10-11% higher during the time trial (p < 0.05) after HCC. These data suggest that carbohydrate loading 18 h previously by means of a 2-h HCC improves cycling performance by 3.3% without any change in pattern of substrate oxidation.