Cortisol is a steroid hormone produced by the adrenal glands, playing a role in the stress response, metabolism, and immune function. A cortisol saliva test is a non-invasive way to measure the level of “free” cortisol, which is the biologically active fraction of the hormone not bound to proteins. This method is highly valued because obtaining a saliva sample avoids the stress of a blood draw, which could artificially elevate cortisol levels and skew the result. Test accuracy relies heavily on precise timing, as cortisol naturally fluctuates in a dramatic pattern throughout the day, following the body’s internal clock.
Understanding Cortisol’s Daily Rhythm
Cortisol levels follow a well-established pattern known as the diurnal rhythm, which is the biological rationale for the strict collection schedule. The body is programmed to produce a surge of cortisol shortly after waking, which helps prepare the body for the day’s demands. This rapid rise is called the Cortisol Awakening Response (CAR) and typically results in the day’s highest cortisol measurement, peaking around 30 to 45 minutes after an individual wakes up.
After reaching its peak, cortisol concentration begins a steady, gradual decline throughout the day. The level is expected to fall by approximately 10 to 15 percent every few hours, allowing the body to transition from alertness to rest. The curve continues downward until it reaches its lowest point, or nadir, late in the evening, typically near midnight. Multiple collections are necessary to map this entire rhythm and determine if the pattern is normal or dysregulated.
The Standard Multi-Point Collection Schedule
The standard protocol requires multiple collections, usually four, spread across the day to accurately map the unique diurnal curve of an individual. The first collection is timed to capture the peak of the Cortisol Awakening Response (CAR), and is often collected either immediately upon waking and again 30 minutes later, or just the single sample 30 minutes after waking. This early morning sample establishes the baseline and the body’s initial hormonal response to the start of the day.
The second sample is typically collected around midday, or roughly four to six hours after waking. This point verifies that the cortisol level has dropped significantly from the morning peak. The third collection is scheduled for the late afternoon or early evening, often between 4:00 PM and 8:00 PM. This sample should show a continued decline, indicating that the body is preparing to wind down for the night.
The final collection is taken at night, usually between 10:00 PM and 12:00 AM, just before going to sleep. This late-night sample is meant to capture the lowest point in the cycle, which should be very low in healthy individuals. It is imperative to record the exact collection time for every sample, as the interpretation of the result depends entirely on its correlation with the time of day it was taken relative to the waking time.
Activities That Immediately Affect Collection Timing
To ensure the salivary cortisol measurement accurately reflects the body’s hormone status, several activities must be avoided immediately before collection. Eating or drinking, particularly beverages containing caffeine or alcohol, can acutely influence the hormone level or contaminate the sample. It is generally required to abstain from all food and drinks for at least 30 to 60 minutes prior to collecting the saliva. Rinsing the mouth with water about 10 minutes before collection may be recommended to clear residues.
Oral hygiene practices also interfere with sample integrity. Brushing the teeth, flossing, or using mouthwash must be avoided for at least 15 to 30 minutes before collection. Ingredients in toothpaste and mouthwash can contaminate the sample, and brushing can cause minor gum bleeding, which introduces blood into the saliva and invalidates the test.
Intense physical activity, such as strenuous exercise, can temporarily elevate cortisol levels. Therefore, it is important to avoid significant exertion and ensure a period of relaxation for up to an hour before any scheduled collection time to prevent an artificially inflated result. Certain medications, especially steroid creams or oral supplements, can also interfere with the results, and patients should consult their healthcare provider about any necessary adjustments to their routine before the collection day.