Aim <p>Most data report that women exhibit higher fat oxidation (FOx) than men during exercise, yet studies typically prescribe exercise using percent maximal heart rate or oxygen uptake (%HR/VO₂max) which does not match metabolic strain.</p> Purpose <p>To examine sex differences in substrate metabolism when exercise is prescribed using %HRmax and ventilatory threshold (VT).</p> Methods <p>Active men (n = 24) and women (n = 25) underwent incremental cycling to determine VO₂max, HRmax, peak power output (PPO), and VT. After an overnight fast, participants subsequently completed 35&#xa0;min of cycling at 62%HRmax or 30% below VT.</p> Results <p>At 62%HRmax (workload = 31 ± 6 vs. 24 ± 7%PPO, p &lt; 0.001)<b>,</b> there was no difference in RER (0.88 ± 0.05 vs. 0.85 ± 0.06, p = 0.12) or FOx (4.6 ± 2.3 vs. 4.8 ± 2.0&#xa0;mg/kg FFM/min, p = 0.78) between men and women, although men revealed higher carbohydrate oxidation (CHOOx) versus women (16.4 ± 4.8 vs. 12.9 ± 6.1&#xa0;mg/kg FFM/min, p = 0.03). Cycling at 30% below VT (workload = 46 ± 5 vs. 44 ± 7%PPO, p = 0.20) revealed no difference in RER (0.92 ± 0.06 vs. 0.90 ± 0.05, p = 0.16), FOx (3.9 ± 3.2 vs. 4.8 ± 2.6&#xa0;mg/kg FFM/min, p = 0.49), or CHOOx (27.3 ± 7.8 vs. 24.6 ± 11.1&#xa0;mg/kg FFM/min, p = 0.35).</p> Conclusion <p>Data reveal no sex differences in FOx when exercise is prescribed according to VT or %HRmax.</p>