The salivary alpha amylase (sAA) enzyme or ptyalin is one of the most important enzymes present in human saliva. The ptyalin has multiple functions: it allows digestion to begin in the oral cavity and plays an important role in the modulation of adhesion and bacterial growth on dental surfaces. 1 The sAA activity correlates to the sympathetic-adrenalmedullary axis. For this reason, the measurement of sAA activity was used in the study, to investigate the psychobiology of stress, suggesting the existence of a correlation with various acute and chronic stress-connected diseases. [2][3][4] According to the recent literature, subjects afflicted with chronic pain, among which temporomandibular disorders (TMD), suffer from a dysregulation of the descending systems of pain modulation (and the periaqueductal gray plays a key role) and of the hypothalamic-pituitary-adrenal axis (HPA) that can be evaluated through the study of the plasmatic cortisol. [5][6][7] Plasmatic cortisol levels are correlated with the salivary cortisol and sAA levels. 8 Cortisol is the principal circulating glucocorticoid released by adrenal glands, controlled by pituitary adrenocorticotropic hormone (ACTH) and hypothalamic corticotropin releasing hormone (CRH). Moreover, the interaction between CRH, endorphin system, and the opposite effect on pain modulation at the central level were demonstrated. 9,10 Transcutaneous electrical nerve stimulation (TENS) was used for a long time to relieve pain. [11][12][13] It is possible that the main effect acts through modulating descending influence from the ventral-lateral periaqueductal gray (PAG) to the rostroventral medial medulla. In particular, low-frequency TENS seems to activate µ opioid receptors of rostral ventromedial medulla TENS effects on salivary stress markers: A pilot study E Ortu, D Pietropaoli, G Mazzei, R Cattaneo, M Giannoni and A Monaco Abstract Transcutaneous electrical nerve stimulation (TENS) is extensively used as pain relief through endorphins release. Moreover, recent findings showed a role in the activation of the autonomic nervous system (ANS); it was evidenced by modification in the heart rate variability and ANS-related marker. The objective of this pilot study is to evaluate salivary alpha amylase (sAA) as a marker of stress in two groups of healthy subjects, one receiving ultra-low frequency transcutaneous electrical nerve stimulation (ULF-TENS) and one without stimulation. Sixty healthy people were enrolled. The test group consisted of 30 participants (15 men, 15 women). The control group consisted of 30 participants (15 men, 15 women). Statistical analysis showed that sAA levels were statistically different between men and women independently from TENS; we hypothesize that treatment could influence sAA levels because it is thought to activate μ opioid receptors. The results of this study seem to indicate that the analysis of sAA, through a non-invasive saliva sample, could be an efficient aid for understanding the functions of the autonomic nervous system.