This study investigated whether physiological demand or gait mechanics differ between sexes during treadmill load carriage. Female (n = 15) and male (n = 15) military recruit-type participants with no load carriage experience completed three 10-minute walking trials at a self-selected speed with increasing relative body-borne loads (0%, 20%, and 40% body weight). A range of cardiorespiratory, perceptual and biomechanical variables were measured. Self-selected walking speed was similar between sexes (4.6-4.8 km·h-1, p > .05) and there were no significant sex-by-load interactions for any variables. Absolute VO2 and VCO2 were greater in males (difference 175-178 mL·min-1, p < .001), however, when relative to body mass, VO2 was similar between sexes (p > .05). Across all loads, cadence was 7 ± 2 steps·min-1 faster (p = .004) and stance time was 0.06 ± 0.02 s shorter (p = .013) in females. Increasing load resulted in greater physiological demand, cadence, % stance time, and step length (p < .05). Practitioner summary: Literature comparing physiological and biomechanical variables between sexes during load carriage is scarce. Physiological and biomechanical sex differences were limited to relative measures associated with physical size (height and mass). Future research may pool male and female participants when conducting trials up to ten minutes in length. Abbreviations: BW: body weight; COM: centre of mass; HR: heart rate; HRmax: maximum heart rate; RER: respiratory exchange ratio; RPE: rating of perceived exertion; VCO2: volume of carbon dioxide; VE: ventilation; VO2: volume of oxygen; VO2max: maximum volume of oxygen.