During aerobic exercise, utilization of carbohydrates (CHO) and fat in exercising muscles, can be influenced by intrinsic biochemical factors, as well as extrinsic factors, such as exercise modality (Edgan et al., 2016). Substrate metabolism has previously been shown to be different between modes of exercise, specifically during running and cycling (Achten et al., 2003; Chenevière et al., 2010). Whether substrate oxidation differs across other exercise modalities remain unclear. This study investigated the effects of treadmill, elliptical and rowing exercise on fat and CHO oxidation, maximal fat oxidation rates (MFO), the exercise intensities at which they occur (Fatmax), and the crossover point where the energy contribution of fat and CHO were equal. Nine males (22 ± 0.5 years, 77.5 ± 3.71 kg, 178.2 ± 2.94 cm) participated in three adapted maximal oxygen consumption tests on a treadmill, elliptical and rowing ergometer. Indirect calorimetry was used to assess substrate oxidation variables during exercise. Venous blood samples were used to determine changes in blood gases, lactate, and pH. Multiple linear mixed-effect regression analyses were performed to assess differences in substrate oxidation curves. Two-way RM ANOVA were used to assess differences in blood gases across conditions, before and after exercise. Maximal fat oxidation rates were significantly higher with the treadmill (0.611 ± 0.0552 g.min-1) compared to both the elliptical (p=0.022, 0.41 ± 0.078 g.min-1) and the rower (p=0.017, 0.40 ± 0.078 g.min-1). Fatmax values were significantly higher with the treadmill (56 ± 6.2 % VO2peak) compared to both the elliptical (p=0.049, 37 ± 5.4 %VO2peak) and rower (p=0.021, 32 ± 5.0 %VO2peak). Mean crossover points were not different between exercise conditions (p=0.388). Lactate concentration was higher in rower (17.39 ± 2.79 mmol.L-1) vs. treadmill after exercise (p=0.021, 13.09 ± 2.26 mmol.L-1), while partial pressure of O2 was lower in rower (37.7 ± 10.8 mmHg) vs. treadmill after exercise (p=0.007, 44.2 ± 19.9 mmHg). No differences were observed between conditions for blood pH. Regression analyses revealed that the fat but not CHO oxidation curve with treadmill was different compared to rower (p<0.001). Exercise on the treadmill maximizes fat oxidation to a greater extent than elliptical and rowing exercises. The treadmill remains a modality that can be used during exercise to help improve cellular and metabolic health. Future investigations should investigate the influence of exercise modality in metabolically-diseased populations.