Introduction This study investigated the effects of body weight (BW) reduction and hypoxia on physiological and perceptual responses during high-intensity interval exercise (HIIE) on the anti-gravity AlterG® treadmill. Material & methods Twenty-six participants (12 women, age: 26.2 years, height: 170.4 cm, weight: 67.8 kg, VO2max: 61.1 mL/min/kg) completed a HIIE in 5 randomized conditions: normoxia at 100%BW; normoxia at 80%BW; normoxia at 60%BW; hypoxia (FIO2 = 0.14) at 80%BW; and hypoxia at 60%BW. The HIIE included 3 sets of 8 × 30-s efforts interspersed with 30-s rest at 110% peak treadmill speed. Heart rate (HR), pulse arterial O2 saturation (SpO2), muscle deoxyhemoglobin concentration ([HHb]), and rate of perceived exertion (RPE) were continuously recorded. Blood lactate concentration ([Lac−]) was measured post-session. Results BW reduction decreased HR, [Lac−] and RPE compared to control (p textless 0.05) and [HHb] in men at the lowest %BW. When hypoxia was added, SpO2 was reduced from 98% to 85%. HR remained lower in all conditions compared to control (p textless 0.05). RPE and [HHb] were higher in hypoxia than normoxic equivalent conditions (p textless 0.05). [Lac−] was higher in Hyp80% compared to other conditions for men (p textless 0.05). Despite subtle differences, men and women responded similarly to this exercise–environment combination. Conclusion Hypoxia effectively restored physiological stress during HIIE despite BW reduction, primarily impacting systemic rather than local muscular physiological parameters. This combination of methods may be beneficial in the rehabilitation and performance context.