The objective of this study was to evaluate the biomaterial properties of nano-modified surface acupuncture needles and the effect of such needles on human pain perception by using pressure pain threshold (PPT) measurements. It is known that changing a material’s surface nano-topography or nanostructure has strong effects on its physical, chemical, and biological surface properties. However, there is no information in the literature about the stimulation characteristics of acupuncture needles with nano-topography or nanostructured surfaces. Based on the knowledge on nanostructured surfaces, it may be possible to potentiate the effects of acupuncture needle stimulation. The pressure pain sensitivity of the masseter muscle in the orofacial region was studied in 21 healthy volunteers in two randomized, double-blinded sessions: an active session of manual acupuncture manipulation with nano-modified surface needles, and an inactive session of sham acupuncture stimulation to control for possible placebo effects. Three acupuncture points were selected from classical Chinese medicine literature: LI4 (Hegu) on the hand, ST6 (Jiache) on the lower masseter region, and ST7 (Xiaguan) on the upper masseter region. PPT measurements, perceived sensations, and pain from the acupuncture were recorded. The results showed discrete yet significant differences in PPT values between the active and inactive acupuncture treatments and significantly higher pain scores from active acupuncture stimulation than from sham acupuncture. These results indicate subtle but significant effects of acupuncture stimulation with nano-modified surface needles, compared to sham acupuncture in healthy participants.