We investigated how the presentation and the manipulation of an optical flow while running on a treadmill affect perceived locomotor speed (Experiment 1) and gait parameters (Experiment 2). In Experiment 1, 12 healthy participants were instructed to run at an imposed speed and to focus on their sensorimotor sensations to be able to reproduce this running speed later. After a pause, they had to retrieve the reference locomotor speed by manipulating the treadmill speed while being presented with different optical flow conditions, namely no optical flow or a matching/slower/faster optical flow. In Experiment 2, 20 healthy participants ran at a previously self-selected constant speed while being presented with different optical flow conditions (see Experiment 1). The results did not show any effect of the presence and manipulation of the optical flow either on perceived locomotor speed or on the biomechanics of treadmill running. Specifically, the ability to retrieve the reference locomotor speed was similar for all optical flow conditions. Manipulating the speed of the optical flow did not affect the spatiotemporal gait parameters and also failed to affect the treadmill running accommodation process. Nevertheless, the virtual reality conditions affected the heart rate of the participants but without affecting perceived effort.