This study examined whether a high-intensity interval (IT) or a continuous steady-state (CT) exercise training program had the greatest effect on fat oxidation rates and fat mass loss in a population of untrained overweight and obese females. Thirteen female subjects (VO2peak 30.6 ± 1.29 ml.kg.min-1, BMI 29 ± 0.79, fat mass [FM] 33.3 ± 2.09 kg) were randomly assigned to either a CT (exercise at the relative intensity that elicits the maximal fat oxidation rate [FATmax] ) or an IT (intervals alternating 5 minutes at 40% and 85% VO2peak) training group that exercised approximately 1 hour, 3 days.week-1 for 10 weeks. Body composition assessments, peak oxygen uptake (VO2peak), FATmax and plasma free fatty acid (FFA) concentrations were examined pre- and post-training using dual-energy X-ray absorptiometry (DEXA), ParvoMedics gas analysis system and FFA half micro tests (Roche Diagnostics). No significant differences were found post-training in body weight (kg), body fat (%), fat-free mass, or fat mass (P>0.05). The relative exercise intensity that elicited FATmax was significantly increased from 35.3 ± 2.55% to 44.7 ± 3.56% in the IT group post-training (P <0.05). The maximal fat oxidation rate was determined at a higher relative exercise intensity after 10 weeks of a IT program compared with a CT program, which resulted in longer durations of fat oxidation during submaximal exercise bouts. These data suggest that an IT program induces a greater increase in the relative exercise intensity that elicits maximal fat oxidation after 10 weeks of training compared to a CT program in this population. Although body composition and FATmax were not altered, it is possible that through training induced metabolic adaptations from the IT program, intramuscular triacylglyceride (IMTG) contributions to fat oxidation at a given steady-state work rate could be increased post-training.