Prediabetes is associated with impaired oxidative capacity and altered substrate utilization during exercise. The effects of continuous (CONT) versus interval (INT) exercise training on fat oxidation during an acute exercise bout at the same absolute and relative intensities are unknown in this population. Obese females/males (n = 17, n = 5) with prediabetes (BMI 32.2 ± 1.2 kg·m^-2; age 62.8 ± 1.6 y; fasting glucose 103.4 ± 1.6 mg·dL^-1; 2-hour glucose 153.7 ± 7.1 mg·dL^-1; VO₂peak 19.9 ± 1.0 mL·kg^-1·min^-1) were screened with a 75g OGTT. Subjects completed a peak oxygen consumption test and a submaximal exercise substrate utilization test consisting of 5min stages at absolute (30W) and relative (70%HR_peak) intensities before and after randomization to 12 sessions (60min each) of CONT (70% HR_peak) or INT (alternating 3min 90% HR_peak, 3min 50% HR_peak) over a two-week period. Body mass decreased and VO₂peak increased more after INT than CONT (INT: -0.6 ± 0.2 kg, CONT: -0.1 ± 0.2 kg; p = 0.04; INT: 1.9 ± 0.6 mL/kg/min, CONT: 0.1 ± 0.6 mL·kg^-1·min^-1; p = 0.04). Training increased fat oxidation by 0.7 ± 0.2 mL·kg^-1·min^-1 during the absolute intensity test (p < 0.001), independent of intensity. During the relative intensity test, fat oxidation increased more after INT than CONT (INT: 1.3 ± 0.4 mL·kg^-1·min^-1, CONT: 0.3 ± 0.3 mL·kg^-1·min^-1; p = 0.03), with no difference in exercise energy expenditure between groups. Enhanced fat oxidation during the relative test was correlated with increased VO₂peak (r = 0.53 p = 0.01). High intensity INT training enhances fat oxidation during the same relative intensity exercise in people with prediabetes.

• Interval exercise training augments fat oxidation in adults with prediabetes

• Elevated exercise fat oxidation correlates with improved aerobic fitness

• Fat oxidation changes were not correlated with glucose regulation

• Enhanced fat oxidation and fitness occur before clinically significant weight loss