The periaqueductal gray (PAG) is involved in the descending modulation of defensive behavior and pain through μ, κ, and δ opioid receptors. Transcutaneous electric nerve stimulation (TENS) has been extensively used for pain relief, as low-frequency stimulation is able to activate µ receptors. The aim of the present study is to use ANS polygraph and salivary/serum biomarkers to evaluate the effect of low-frequency TENS stimulation of ANS in TMD patients.