Reliability of three versus five saliva sampling times for assessing the cortisol awakening response

The cortisol awakening response (CAR) describes the sharp increase in cortisol secretion within 60 min after awakening. A summary of the CAR, the area under the cortisol curve above the awakening cortisol value (AUC_i) is a widely used biomarker in health research. Estimation of the AUC_i rely on a number of collected salivary samples at fixed time intervals (i.e., 5 samples in 15 min intervals) starting from awakening. Little empirical work has been executed to investigate the impact of reducing sampling times on AUC_i estimation, which could potentially improve participant compliance and reduce operational costs. This study aimed to assess the reliability and validity of using 3-sample AUC_i versus 5-sample AUC_i, i.e., systematic and random fluctuations based on a large dataset from healthy and case individuals (total n = 537). We showed that the ideal timing of 3-sampling times was 0-30-60 min with a median difference in AUC_i of - 8 nmol*h/L and interquartile range of 65 nmol*h/L among healthy individuals, and - 12 nmol*h/L and 78 nmol*h/L among case individuals. We subsequently validated the 3-sample AUC_i by re-analyzing three published association studies. Overall, we obtained similar p-values with 3-sample AUC_i when compared to 5-sample AUC_i, while smaller effect sizes and standard errors were observed. In conclusion, despite a less precise estimation of the AUC_i itself, our data support that the AUC measure of the CAR, based on three samples collected at 0-30-60 min from awakening, provides reliable results in association studies.