On awakening cortisol should increase about 50% in the first 30 minutes then begin to progressively drop the remainder of the day. Three, rather than one, early morning collections are what is needed to accurately assess the CAR; one immediately on waking, one 30 minutes later, and another at 1 hour.
Diurnal Rhythm Assessment
Thirty minutes after awakening from a good night's sleep, cortisol levels are at the highest they'll be all day. Following the morning peak, cortisol levels then fall to less than half that peak level by noon. They continue to drop to very low levels at night where they stay low during the sleep hours. Some individuals have a sharp rise to reach morning levels, others a more gradual incline. Looking at cortisol levels graphed during the day, any abnormal elevation, or depression of levels, or a loss of the expected curve with its characteristic morning peak and swooping decline towards evening may suggest HPA axis dysfunction – which is what we're most interested in assessing when we're looking at a 4-point salivary cortisol test.