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Studies evaluating mechanical asymmetry across a range of running velocities during treadmill runs have yielded inconsistent findings, while the impact of additional hypoxic exposure has never been investigated. The aim of this study was to characterize the effects of manipulating running velocity and hypoxic exposure on gait asymmetry during treadmill running. Eleven trained individuals performed seven runs at different velocities (8, 10, 12, 14, 16, 18, and 20 km·h−1) in a randomized order, each lasting 45 s. The running took place on an instrumented treadmill for normoxia (FiO2 = 20.9%), moderate hypoxia (FiO2 = 16.1%), high hypoxia (FiO2 = 14.1%), and severe hypoxia (FiO2 = 13.0%). Vertical and antero-posterior ground reaction force recordings over 20 consecutive steps (i.e., after running ∼25 s) allowed the measurement of running mechanics. Lower-limb asymmetry was assessed from the ‘symmetry angle’ (SA) score. Two-way repeated-measures ANOVA (seven velocities × four conditions) was used. There was no significant difference in SA scores for any of the biomechanical variables for velocity (except contact time and braking phase duration; p = 0.003 and p = 0.002, respectively), condition, or interaction. Mean SA scores varied between ∼1% and 2% for contact time (1.5 ± 0.8%), flight time (1.6 ± 0.6%), step length (0.8 ± 0.2%), peak vertical force (1.2 ± 0.5%), and mean vertical loading rate (2.1 ± 1.0%). Mean SA scores ranged from ∼2% to 5% for duration of braking (1.6 ± 0.7%) and push-off phases (1.9 ± 0.6%), as well as peak braking (5.0 ± 1.9%) and push-off forces (4.8 ± 1.7%). In conclusion, the trained runners exhibited relatively even strides, with mechanical asymmetries remaining low-to-moderate across a range of submaximal, constant running velocities (ranging from 8 to 20 km·h−1) and varying levels of hypoxia severity (between normoxia and severe hypoxia).