Acute pain is one of the most common complaints concerning referral to the emergency department. Pain remains misdiagnosed and mistreated despite all current pain evaluation measurements (pain scales, physiological parameters). Cortisol is identified as stress and pain-marker during acute pain. The aim of this study was to evaluate any changes in cortisol and if there were any changes during the pain stimulus experiment in healthy volunteers. Methods: a prospective experimental study was performed, and healthy volunteers were included in the study. All volunteers underwent a specific controlled-pain experimental procedure. The first phase (10min) – calm sitting followed by cold-water stimulus (2min, left hand, 7℃ water); recovery phase (5min) – calm sitting followed by a second cold-water stimulus (2min, left hand, 10℃ water); recovery phase (10min) followed with a deep breathing (1min) phase and 5min calm sitting. Saliva samples were collected at 8 time points as follows: at 0 (1) 10 (2), 12 (3), 17 (4), 19 (5), 29 (6), 30 (7), and 35min (8) respectivelly. All samples were stored at -80℃ until tested with the cortisol ELISA kit. Statistical analysis was performed using SPSS 27.0; p-value