The use of mixed reality (MR) has been explored in many areas of human-machine interfacing (HMI) such as vehicles, assistive devices and medical equipment. It is reported that cyber sickness, like motion sickness, in virtual environments is caused by a conflict between moving visual stimulation and body movement. We have investigated how to reduce the cyber sickness for a driver assistance system using a MR display with a head-up display (HUD). In this study, we evaluated the cyber sickness caused by a spatio-temporal difference between the virtual image and the real image while subjects are moved. In the movement, the pitch sinusoidal motion is varied in the range from 0.1 to 2.0 Hz using a driving simulator with the MR display. The evaluations were performed by measuring the visual functions (dark focus and accommodation), equilibrium functions (COP length and area), autonomous nervous system (respiratory and sweating rate) and the subjective reports regarding stress and fatigue. The results showed that no significant influences were observed at the high and low frequencies of the motion during these evaluation tests. However, at the middle frequency (about 0.2 Hz), the spatio-temporal difference apparently caused a postural instability and an increase of peak respiratory frequency and variability in sweating rate. Finally, we propose the hypothesis of biological effect induced by spatio-temporal discrepancy in driver assistance systems that use MR displays.