Salivary Cortisol Awakening Response Measurement

Salivary cortisol was collected at three time-points to determine the cortisol awakening response (CAR): immediately after awakening and 30 and 45min thereafter (Powell and Schlotz, 2012 (link)). All samples were then stored at 4°C until sent to the laboratory. Once in the laboratory, saliva samples were centrifuged for 5-min at 2000 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 were 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 (AUCg) and the area under the curve with respect to increase (AUCi) (Pruessner et al., 2003 (link)).