Virtual reality (VR) is an emerging technology offering tremendous opportunities to aid gait rehabilitation. To this date, real walking with users immersed in virtual environments with head-mounted displays (HMDs) is either possible with treadmills or room-scale (overground) VR setups. Especially for the latter, there is a growing interest in applications for interactive gait training as they could allow for more self-paced and natural walking. This study investigated if walking in an overground VR environment has relevant effects on 3D gait biomechanics. A convenience sample of 21 healthy individuals underwent standard 3D gait analysis during four randomly assigned walking conditions: the real laboratory (RLab), a virtual laboratory resembling the real world (VRLab), a small version of the VRlab (VRLab−), and a version which is twice as long as the VRlab (VRLab+). To immerse the participants in the virtual environment we used a VR-HMD, which was operated wireless and calibrated in a way that the virtual labs would match the real-world. Walking speed and a single measure of gait kinematic variability (GaitSD) served as primary outcomes next to standard spatio-temporal parameters, their coefficients of variant (CV%), kinematics, and kinetics. Briefly described, participants demonstrated a slower walking pattern (−0.09 ± 0.06 m/s) and small accompanying kinematic and kinetic changes. Participants also showed a markedly increased gait variability in lower extremity gait kinematics and spatio-temporal parameters. No differences were found between walking in VRLab+ vs. VRLab−. Most of the kinematic and kinetic differences were too small to be regarded as relevant, but increased kinematic variability (+57%) along with increased percent double support time (+4%), and increased step width variability (+38%) indicate gait adaptions toward a more conservative or cautious gait due to instability induced by the VR environment. We suggest considering these effects in the design of VR-based overground training devices. Our study lays the foundation for upcoming developments in the field of VR-assisted gait rehabilitation as it describes how VR in overground walking scenarios impacts our gait pattern. This information is of high relevance when one wants to develop purposeful rehabilitation tools.