In order to keep the standardization related with circadian rhythms saliva samples were collected between 8-9 am. Nevertheless, it was convened 12 am (pre-stress) and between 2-3 pm (post-stress/after unaware exam hour) to evaluate its relationship with stress. Within 60 minutes before the test, individuals were not allowed to smoke, eat, drink liquids or brush their teeth They were asked to soak the swab with saliva for 1 or 2 minutes until they are saturated completely. Tubes were waited 30 minutes at room temperature and then centrifuged for 10-15 minutes at approximately 3200 g. After centrifugation, the swab and small insert were thrown away, and the large outer tube was stored at -80 °C until analysis.
The twenty-three healthy young adult of faculty of dentistry students were recruited to the study. Exclusion criteria were presence of any disease that can affect hypothalamic-pituitary-adrenal (HPA) axis.