This study explored lifestyle and biological determinants of peak fat oxidation (PFO) during cycle ergometry, using duplicate measures to account for day-to-day variation. Seventy-three healthy adults (age range: 19–63 years; peak oxygen consumption [˙VO2peak]:42.4[10.1]ml⋅kgBM−1⋅min−1; n = 32 women]) completed trials 7–28 days apart that assessed resting metabolic rate, a resting venous blood sample, and PFO by indirect calorimetry during an incremental cycling test. Habitual physical activity (combined heart rate accelerometer) and dietary intake (weighed record) were assessed before the first trial. Body composition was assessed 2–7 days after the second identical trial by dual-energy X-ray absorptiometry scan. Multiple linear regressions were performed to identify determinants of PFO (mean of two cycle tests). A total variance of 79% in absolute PFO (g·min−1) was explained with positive coefficients for ˙VO2peak(strongest predictor), FATmax (i.e the % of ˙VO2peak that PFO occurred at), and resting fat oxidation rate (g·min−1), and negative coefficients for body fat mass (kg) and habitual physical activity level. When expressed relative to fat-free mass, 64% of variance in PFO was explained: positive coefficients for FATmax (strongest predictor), ˙VO2peak, and resting fat oxidation rate, and negative coefficients for male sex and fat mass. This duplicate design revealed that biological and lifestyle factors explain a large proportion of variance in PFO during incremental cycling. After accounting for day-to-day variation in PFO, ˙VO2peak and FATmaxwere strong and consistent predictors of PFO.