This study compared physiological and biomechanical responses between treadmill and overground load carriage. Thirty adults completed six 10-minute walking trials across three loads (0%, 20%, and 40% body mass) and two surfaces (treadmill and overground). Relative oxygen consumption was significantly greater on the treadmill for 20% (1.54 ± 0.20 mL⋅kg-1⋅min-1) and 40% loads (1.08 ± 0.20 mL⋅kg-1⋅min-1). All other physiological and perceptual responses were significantly higher in the treadmill condition and with increases in load. Stance time was longer (0%: 0.05s; 20%: 0.02s, 40%: 0.05s, p < 0.001) and cadence was lower (0%: 1 step·min-1; 20%: 2 steps·min-1; 40%: 3 steps·min-1, p < 0.05) on the treadmill. Peak lower limb joint angles were similar between surfaces except for ankle plantarflexion, which was 8˚ greater on the treadmill. The physiological responses to treadmill-based load carriage are generally not transferable to overground load carriage and caution must be taken when conducting treadmill-based load carriage research to inform operational-based scenarios. Practitioner Summary: Literature is limited when comparing the physiological and biomechanical responses to treadmill and overground load carriage. Using a repeated measures design, it was shown that although walking kinematics are generally similar between surfaces, there was a greater physiological demand while carrying a load on a treadmill when compared with overground.