ABSTRACT Acute physiological, perceptual and biomechanical consequences of manipulating both exercise intensity and hypoxic exposure during treadmill running were determined. On separate days, eleven trained individuals ran for 45 s (separated by 135 s of rest) on an instrumented treadmill at seven running speeds (8, 10, 12, 14, 16, 18 and 20 km.h−1) in normoxia (NM, FiO2 = 20.9%), moderate hypoxia (MH, FiO2 = 16.1%), high hypoxia (HH, FiO2 = 14.1%) and severe hypoxia (SH, FiO2 = 13.0%). Running mechanics were collected over 20 consecutive steps (i.e. after running ∼25 s), with concurrent assessment of physiological (heart rate and arterial oxygen saturation) and perceptual (overall perceived discomfort, difficulty breathing and leg discomfort) responses. Two-way repeated-measures ANOVA (seven speeds × four conditions) were used. There was a speed × condition interaction for heart rate (p = 0.045, ηp2 = 0.22), with lower values in NM, MH and HH compared to SH at 8 km.h−1 (125 ± 12, 125 ± 11, 128 ± 12 vs 132 ± 10 b.min−1). Overall perceived discomfort (8 and 16 km.h−1; p = 0.019 and p = 0.007, ηp2 = 0.21, respectively) and perceived difficulty breathing (all speeds; p = 0.023, ηp2 = 0.37) were greater in SH compared to MH, whereas leg discomfort was not influenced by hypoxic exposure. Minimal difference was observed in the twelve kinetics/kinematics variables with hypoxia (p > 0.122; ηp 2 = 0.19). Running at slower speeds in combination with severe hypoxia elevates physiological and perceptual responses without a corresponding increase in ground reaction forces. Highlights The extent to which manipulating hypoxia severity (between normoxia and severe hypoxia) and running speed (from 8 to 20 km.h−1) influence acute physiological and perceptual responses, as well as kinetic and kinematic adjustments during treadmill running was determined. Running at slower speeds in combination with severe hypoxia elevates heart rate, while this effect was not apparent at faster speeds. Arterial oxygen saturation was increasingly lower as running speed and hypoxic severity increased. Overall perceived discomfort (8 and 16 km.h−1) and perceived difficulty breathing (all speeds) were lower in moderate hypoxia than in severe hypoxia, whereas leg discomfort remained unchanged with hypoxic exposure.