Our aim was to examine the effects of seven high-intensity aerobic interval training (HIIT) sessions over 2 wk on skeletal muscle fuel content, mitochondrial enzyme activities, fatty acid transport proteins, peak O2 consumption (V̇o2 peak), and whole body metabolic, hormonal, and cardiovascular responses to exercise. Eight women (22.1 ± 0.2 yr old, 65.0 ± 2.2 kg body wt, 2.36 ± 0.24 l/min V̇o2 peak) performed a V̇o2 peak test and a 60-min cycling trial at ∼60% V̇o2 peak before and after training. Each session consisted of ten 4-min bouts at ∼90% V̇o2 peak with 2 min of rest between intervals. Training increased V̇o2 peak by 13%. After HIIT, plasma epinephrine and heart rate were lower during the final 30 min of the 60-min cycling trial at ∼60% pretraining V̇o2 peak. Exercise whole body fat oxidation increased by 36% (from 15.0 ± 2.4 to 20.4 ± 2.5 g) after HIIT. Resting muscle glycogen and triacylglycerol contents were unaffected by HIIT, but net glycogen use was reduced during the posttraining 60-min cycling trial. HIIT significantly increased muscle mitochondrial β-hydroxyacyl-CoA dehydrogenase (15.44 ± 1.57 and 20.35 ± 1.40 mmol·min−1·kg wet mass−1 before and after training, respectively) and citrate synthase (24.45 ± 1.89 and 29.31 ± 1.64 mmol·min−1·kg wet mass−1 before and after training, respectively) maximal activities by 32% and 20%, while cytoplasmic hormone-sensitive lipase protein content was not significantly increased. Total muscle plasma membrane fatty acid-binding protein content increased significantly (25%), whereas fatty acid translocase/CD36 content was unaffected after HIIT. In summary, seven sessions of HIIT over 2 wk induced marked increases in whole body and skeletal muscle capacity for fatty acid oxidation during exercise in moderately active women.