Two-point discrimination (TPD) has been widely used as a parameter for the examination of higher-order perceptual functions in the field of rehabilitation. Previous research has shown that the threshold of TPD increases with aging or pathological conditions such as stroke or chronic pain. It has also been reported that the threshold can be decreased by continuous tactile or electrical stimulation. The cognitive process in the cortex has been shown to be involved in the determination of the TPD threshold. However, the reliability of TPD has been questioned, because differences in the firing rate of the responding receptors and afferent fibers occur, depending on how the measuring instrument is applied. To investigate the influence of the stimulus condition on the TPD threshold, we utilized a computer-controlled two-point tactile stimulator and measured the TPD threshold by alternating stimulus speed and stimulus penetration depths. We found that a stimulus speed of 5.0 mm/s or 10.0 mm/s and a stimulus penetration depth of 1.0 mm were the optimum condition for measurement, at which the TPD threshold becomes lowest. We also found that no influence is exerted on the threshold by repeated measurement under the stimulus conditions utilized in this experiment. Our findings suggest that TPD measurement should be performed under certain stimulus conditions, as identified in this present study, to obtain reliable results that reflect the highest ability of the subject for spatial discrimination.