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 (AUCi) is a widely used biomarker in health research. Estimation of the AUCi 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 AUCi estimation, which could potentially improve participant compliance and reduce operational costs. This study aimed to assess the reliability and validity of using 3-sample AUCi versus 5-sample AUCi, 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 AUCi 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 AUCi by re-analyzing three published association studies. Overall, we obtained similar p-values with 3-sample AUCi when compared to 5-sample AUCi, while smaller effect sizes and standard errors were observed. In conclusion, despite a less precise estimation of the AUCi 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.