Salivary Cortisol Awakening Response Assessment

Salivary cortisol was collected at three time points to determine the cortisol awakening response (CAR): immediately after awakening and 30 and 45 min thereafter (Powell and Schlotz, 2012 (link)). All samples were stored at 4°C until sent to the laboratory, where they were then centrifuged for 5 min at 2,000 rpm to extract saliva with low viscosity and subsequently transferred to 1.5-ml tubes and stored in a freezer at –80°C until analysis. After thawing, 20 μl of salivary samples was transferred to a sample cup and cortisol was estimated using a cortisol RP Elecsys kit (Roche Diagnostics, United States) in a cobas e 411 analyzer. Summary indexes of CAR included the area under the curve with respect to ground (AUC_G) and the area under the curve with respect to increase (AUC_I) calculated according to the work of Pruessner et al. (2003) (link).