We evaluated the effect of carbohydrate (CHO) loading on cycling performance that was designed to be similar to the demands of competitive road racing. Seven well-trained cyclists performed two 100-km time trials (TTs) on separate occasions, 3 days after either a CHO-loading (9 g CHO ⋅ kg body mass^{− 1} ⋅ day^{− 1}) or placebo-controlled moderate-CHO diet (6 g CHO ⋅ kg body mass^{− 1} ⋅ day^{− 1}). A CHO breakfast (2 g CHO/kg body mass) was consumed 2 h before each TT, and a CHO drink (1 g CHO ⋅ kg^.body mass^{− 1} ⋅ h^{− 1}) was consumed during the TTs to optimize CHO availability. The 100-km TT was interspersed with four 4-km and five 1-km sprints. CHO loading significantly increased muscle glycogen concentrations (572 ± 107 vs. 485 ± 128 mmol/kg dry wt for CHO loading and placebo, respectively; P < 0.05). Total muscle glycogen utilization did not differ between trials, nor did time to complete the TTs (147.5 ± 10.0 and 149.1 ± 11.0 min; P = 0.4) or the mean power output during the TTs (259 ± 40 and 253 ± 40 W, P = 0.4). This placebo-controlled study shows that CHO loading did not improve performance of a 100-km cycling TT during which CHO was consumed. By preventing any fall in blood glucose concentration, CHO ingestion during exercise may offset any detrimental effects on performance of lower preexercise muscle and liver glycogen concentrations. Alternatively, part of the reported benefit of CHO loading on subsequent athletic performance could have resulted from a placebo effect.