The purpose of the current study was to examine the influence of dietary composition on short-term endurance training-induced adaptations of substrate partitioning and time trial exercise performance. Eight untrained males cycled for 90 min at approximately 54% aerobic capacity while being infused with [6,6(2)H]glucose before and after two 10-d experimental phases separated by a 2-week washout period. Time trial performance was measured after the 90-min exercise trials before and after the 2nd experimental phase. During the first 10-d phase, subjects were randomly assigned to consume either a high carbohydrate or high fat diet while remaining inactive (CHO or FAT). During the second 10-d phase, subjects consumed the opposite diet, and both groups performed identical daily supervised endurance training (CHO+T or FAT+T). CHO and CHO+T did not affect exercise metabolism. FAT reduced glucose flux at the end of exercise, while FAT+T substantially increased whole body lipid oxidation during exercise and reduced glucose flux at the end of exercise. Despite these differences in adaptation of substrate use, training resulted in similar improvements in time trial performance for both groups. We conclude that (a) 10-d high fat diets result in substantial increases in whole body lipid oxidation during exercise when combined with daily aerobic training, and (b) diet does not affect short-term training-induced improvements in high-intensity time trial performance.