The objective of this study was to examine the effects of Virtual Reality (VR) habituation on the kinematic and gait variability changes in older adults. Sixteen healthy older adults participated in the study. The experiment consisted of walking session on a walkway, followed by walking on the treadmill with and without a head mounted display of a VR scene. Kinematic data were collected using a motion capture system. Additionally, a cyber-sickness questionnaire was administered to the participants during and after the experiment to evaluate motion sickness symptoms. Statistical analyses indicated an increased variability in stride length, stride velocity, and step width during the initial 5-10 min in the VR. Kinematic data indicated an increased ankle plantar flexion, knee flexion, and trunk flexion in the initial VR period. All parameters approximated normal treadmill walking within 20 min of VR walking. The cyber sickness scores indicated no presence of cyber sickness during or after the VR experiment. It was concluded that the habituation time in VR may have significant effect on gait behavior in older adults and therefore it should be considered while designing a VR locomotion training study.