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%: 0.05 s; 20%: 0.02 s, 40%: 0.05 s, 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 plantar flexion, 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. Abbreviations: BM: body mass; e.g: for example; HR: heart rate; HR max : heart rate maximum; Hz: hertz; kg: kilograms; km·h −1 : kilometres per hour; L⋅min −1 : litres per minute; m: metres; MD: mean difference; mL·kg −1 ·min −1 : millilitres per kilogram per minute; mL⋅min −1 : millilitres per minute; η 2 p: partial-eta squared; OG: overground; RPE: rating of perceived exertion; s: seconds; SD: standard deviation; SE: standard error; steps·min −1 : steps per minute; TM: treadmill; V̇CO 2 : volume of carbon dioxide; V̇E: ventilation; V̇O 2 : volume of oxygen; V̇O 2max : maximum volume of oxygen; y: years