We examined the effect of acupuncture treatment in the supine position, sitting position, and horseback riding posture on a horseback riding simulator at a standstill from the aspects of physical movement, psychological assessment, and autonomic nerve activity. Twenty-nine university students suffering from pain and stiffness during lateral hip flexion and extension movement of the Straight Leg Raising and movement of the neck and shoulders were randomly assigned to the supine position, sitting position, and horseback riding posture, and a tape-type press needle was attached at four acupuncture points on both lower limbs. Regarding the measuring method, we conducted M-Test movement assessment, two-dimensional mood standard (TDMS-ST), and heart rate variability analysis. There was no difference in the baseline among the three groups prior to the experiment and the improvement in the movement of the neck, SLR, and hip flexion (knee flexed position) for all three groups along with the comfort level of TDMS-ST significantly increased following treatment. Moreover, while we also observed a significant increase in HF (parasympathetic activity index) for all three groups following treatment, no change was observed in the LF/HF ratio (sympathetic activity index). These findings suggest that the stimulation from the press needle by the M-Test method achieved a relaxing effect regardless of the posture because the stimulation had a low degree of invasiveness, and it was also not associated with any pain.