The Essentials of Cortisol Saliva Test You Must Know

Cortisol test display on tablet

What is cortisol and why is it important to test it?

Cortisol is a steroid hormone produced by the adrenal glands that plays a vital role in regulating metabolism, immune function, blood pressure, and the body's response to stress. It is often referred to as the "stress hormone" since levels fluctuate in response to physical or psychological stress. Cortisol levels follow a circadian rhythm and are typically highest in the morning after waking up and lowest in the evening before bedtime.

Abnormal cortisol levels can indicate disorders of the hypothalamic-pituitary-adrenal (HPA) axis like Cushing's syndrome or Addison's disease. Cushing's syndrome is characterized by excessive cortisol production while Addison's disease is caused by adrenal insufficiency and insufficient cortisol. Testing cortisol levels helps diagnose these conditions and monitor treatment effectiveness. Cortisol testing may use blood, urine, or saliva samples. Salivary cortisol testing is emerging as a preferred method for assessing cortisol rhythms and diagnosing adrenal disorders.

How is the salivary cortisol test performed and what are the sample requirements?

The salivary cortisol test measures the level of free unbound cortisol in saliva, which closely correlates with levels of free serum cortisol. Cortisol passes from the bloodstream into saliva by diffusing through the salivary glands. Salivary cortisol reflects the biological active form of cortisol and is not affected by conditions that alter cortisol protein binding.

Patients are typically provided a commercial saliva collection kit containing tubes, collection swabs, and detailed instructions. Common collection times are first morning upon waking, 30-60 minutes after waking, midday, evening, and late night before bed. The patient places the absorbent swab under the tongue for 1-2 minutes until saturated with saliva. The swab is then placed back in the tube without touching it. Tubes are labeled with patient information and collection time. Patients refrigerate samples until returning to the lab.

At least 0.5-1 mL of clear saliva free of blood or particulates is required. Samples are stable refrigerated for up to a week and frozen for longer periods. Food intake, brushing teeth, smoking, and blood contamination can affect results. Patients should avoid these 30-60 minutes before collecting saliva.

What are normal cortisol ranges throughout the day?

In healthy individuals, salivary cortisol follows a distinct circadian rhythm with peak levels in the early morning up to 20-25 nmol/L and a nadir at midnight of <3.5 nmol/L. Levels then rise steeply in the first hour after waking, peaking around 30-45 minutes after waking at 10-15 nmol/L. This post-waking spike is absent in adrenal insufficiency. Levels gradually decline throughout the day reaching 3-10 nmol/L in the evening.

Reference ranges vary by laboratory but typical norms are:

✅Morning: 10-25 nmol/L
✅Afternoon: 5-10 nmol/L
✅Evening: <10 nmol/L
✅Night: <3.5 nmol/L

Abnormal loss of diurnal variation or cortisol levels outside expected ranges can indicate medical conditions affecting the HPA axis.

How does salivary testing help diagnose Cushing’s syndrome?

Elevated nighttime salivary cortisol is a useful screening test for Cushing's syndrome, with midnight samples >145 ng/dL (4 nmol/L) suggesting Cushing's. Midnight salivary cortisol assays have a sensitivity of 96-100% and specificity of 93-100% for Cushing’s. The test is convenient, non-invasive, and avoids hospitalization for midnight serum sampling.

If midnight salivary cortisol is high, confirmatory testing is warranted such as a 24-hour urinary free cortisol, dexamethasone suppression test, or ACTH stimulation test. Salivary testing may also be used to monitor treatment effectiveness in Cushing’s patients. With successful treatment, abnormally high midnight salivary cortisol levels should decrease to normal.

How does the test help diagnose Addison’s disease?

Patients with Addison's disease fail to produce the early morning cortisol spike and high levels. First morning and 30-60 minute post-waking salivary cortisol are typically low. midnight levels may also be low as the normal late-night rise in ACTH drives minimal cortisol secretion in adrenal insufficiency.

The 30-60 minute post-waking salivary cortisol test has a sensitivity and specificity exceeding 90% for diagnosing Addison’s disease. Critics argue the test's accuracy depends on patient adherence with timing and protocols. To compensate, some recommend additional sampling times or combining the first morning sample with a cosyntropin stimulation test.

What are the advantages of salivary over serum cortisol testing?

Several characteristics make salivary cortisol an attractive alternative to traditional serum testing:

✅Non-invasive collection
✅Convenient for assessing circadian rhythms with multiple sampling
✅Avoids hospitalization for late-night blood draws
✅Reflects free biologically active cortisol levels
✅Easy sample storage and transport
✅Can be used in field studies

Salivary results can be impacted by improper collection technique, blood contamination, and adherence to timing protocols. Optimal diagnostic accuracy requires close patient cooperation. Providers should give clear instructions on collecting samples.

What is the clinical utility of salivary cortisol testing?

Applications of salivary cortisol testing include:

✅Screening for Cushing’s syndrome - elevated nighttime levels
✅Diagnosing Addison's disease - blunted morning spike
✅Monitoring treatment effectiveness in adrenal disorders
✅Assessing recovery of the HPA axis after long-term steroid use
✅Evaluating cortisol rhythms in research studies on stress, fatigue, depression, and other conditions
✅Detecting abnormal cortisol responses to acute stress
✅Monitoring cortisol in newborns and children where blood draws are difficult

Clinical interpretation of salivary cortisol requires an understanding of the HPA axis and influences on cortisol secretion. Providers should consider the patient’s symptoms and clinical context when interpreting results. An endocrinologist may be consulted for equivocal findings.

What are possible limitations or drawbacks of the test?

Potential limitations of salivary cortisol testing include:

✅Results altered by improper collection, handling, and storage
✅Non-adherence to timing protocols and collection instructions
✅Inability to obtain samples in young children
✅Lack of established reference ranges across laboratories
✅Interference from traces of blood or particulates
✅Effects of eating, smoking, and oral health prior to sampling
✅Not recommended in pregnancy due to altered cortisol rhythms

To optimize accuracy, patients should be carefully instructed on proper technique. Samples with visual contamination or improper collection should be rejected. Providers must account for factors that transiently increase cortisol like stress or exercise when interpreting results. Overall, salivary cortisol offers a convenient outpatient method for assessing adrenal function.

Click to View → Mantacc Saliva Collection Tools

References

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2. Kalman BA, Grahn RE. Measuring salivary cortisol in the behavioral neuroscience laboratory. J Undergrad Neurosci Educ. 2004 Spring;2(2):A41-9. Epub 2004 Jun 15. PMID: 23493518; PMCID: PMC3592595.
3. Pritchard BT, Stanton W, Lord R, Petocz P, Pepping GJ. Factors Affecting Measurement of Salivary Cortisol and Secretory Immunoglobulin A in Field Studies of Athletes. Front Endocrinol (Lausanne). 2017 Jul 24;8:168. doi: 10.3389/fendo.2017.00168. PMID: 28790976; PMCID: PMC5522838.