A cortisol test helps to diagnose Cushing syndrome, where there is excess cortisol levels in the body, and to diagnose adrenal insufficiency (Addison disease) where there is deficient cortisol. Cortisol is a hormone and it plays important role in the metabolism of proteins, lipids, and carbohydrates, etc. Normally, cortisol levels show diurnal variations (increase early in the morning, decline throughout the day and reach lowest levels at midnight). Cortisol is produced by the adrenal glands. Its production is regulated by the hypothalamus and the pituitary gland. When the cortisol levels decrease in the blood, hypothalamus releases corticotropin-releasing hormone, which stimulates the production of adrenocorticotropic hormone (ACTH) by pituitary gland. ACTH stimulates the adrenal glands for production and release of cortisol. Cortisol in the blood is mostly bound to a protein (albumin), only a small amount of it is left free. The free form of cortisol is biologically active. Blood cortisol testing evaluates both protein-bound and free cortisol where as urine and saliva testing evaluate only the free cortisol levels. Blood and/or saliva samples which are collected at different times (usually at 8 am and 4 pm) can be used to evaluate both cortisol levels and the diurnal variation of cortisol levels. Unlike serum cortisol, urine cortisol sample will not show diurnal variation but it will measure the total amount of free cortisol that is excreted in 24 hours. If abnormal levels of cortisol are detected, additional tests are done to confirm the diagnosis and to determine the cause. Testing for Excess Cortisol Production: If blood cortisol levels are high, additional tests are done to confirm if the cortisol is truly elevated and to rule out stress-related increase or use of cortisol like medication. The additional testing include measuring of 24-hour urinary cortisol, overnight dexamethasone suppression test, etc. Urinary cortisol requires the collection of urine over 24 hours. This test helps determine if the elevated blood cortisol level represents a true increase, as ACTH is secreted by the pituitary gland in pulses. Dexamethasone suppression test: It involves analyzing a baseline sample for cortisol and then measuring the cortisol levels, at regular intervals after giving oral dexamethasone. Increased levels of cortisol due to stress, decrease the production of cortisol as dexamethasone suppresses ACTH production. Testing for Insufficient Cortisol Production: If adrenal glands are not producing adequate cortisol or if the priliminary blood tests indicate insufficient cortisol production, an ACTH stimulation test may be needed. ACTH stimulation test: Before and after the injection of synthetic ACTH, cortisol levels in the blood are measured. Cortisol levels increase if the adrenal gland is normal and decrease if it is damaged or improperly functioning.