Cortisol awakening response
Morning saliva samples (the morning of the day of study visit) were collected for assessment of the cortisol awakening response using the Salivette system (Sarstedt, Germany). A total of four samples was collected, with the first one collected upon wakening, the second one 30 min later, and the third and fourth one in 15 min increments. Participants were instructed to not brush their teeth until after all saliva samples were collected, to not eat or drink anything prior to the first sample, and to avoid eating and drinking 15 min prior to the remaining samples. Saliva samples were centrifuged at 1500 × Salivary cortisol was analyzed in duplicates using the ENZO Life Sciences enzyme-linked immunosorbent assay kits (Exeter, UK) per manufacturer’s instructions. The lower limit of detection was 0.919 nmol/l. Inter- and intra-assay coefficients of variability were 13.4 % and 10.5%.