Cortisol testing is a key diagnostic tool for healthcare professionals aiming to assess adrenal gland function and the activity of the hypothalamic-pituitary-adrenal (HPA) axis.
This axis plays a fundamental role in regulating the body’s response to stress. Cortisol, commonly referred to as the “stress hormone”, is involved in essential physiological processes, including the regulation of macronutrient metabolism, blood pressure control, modulation of the inflammatory response, and fluid-electrolyte balance.
Below, we discuss the relevance of cortisol testing, optimal timing for sample collection, available testing methods, and result interpretation.
1. Cortisol and its physiological functions
Cortisol is a steroid hormone produced by the adrenal glands in response to stimulation by adrenocorticotropic hormone (ACTH), which is secreted by the pituitary gland. It is essential for the stress response, promoting the mobilisation of energy resources such as glucose, and preparing the body to deal with adverse situations.
Under normal conditions, cortisol follows a circadian release pattern, with high levels in the morning — helping the body to wake up — and lower levels at night, promoting rest.
In addition to its role in the stress response, cortisol performs vital functions such as:
- Regulation of blood glucose and the metabolism of proteins, lipids, and carbohydrates.
- Modulation of inflammation and immune system control.
- Maintenance of blood pressure within healthy range.
- Regulation of fluid and electrolyte balance.
Thus, cortisol is indispensable for maintaining homeostasis, and abnormal levels — whether elevated or deficient — may indicate significant endocrine dysfunctions.
2. Indications for cortisol testing
Cortisol testing is indicated when adrenal or HPA axis dysfunction is suspected, and it is commonly used to diagnose conditions such as Cushing’s syndrome, Addison’s disease, and secondary adrenal insufficiency.
Common signs and symptoms leading to a cortisol test request include:
- Excess cortisol: abdominal weight gain, rounded face, thin skin, easy bruising, muscle weakness, high blood pressure, and psychological symptoms such as anxiety or depression — all suggestive of Cushing’s syndrome.
- Cortisol deficiency: weight loss, extreme fatigue, muscle and joint pain, low blood pressure, and hypoglycaemia, frequently seen in Addison’s disease.
Cortisol testing is also useful for monitoring treatment in cortisol-related disorders or assessing the impact of medications such as corticosteroids.
3. Cortisol testing methods
Which to choose and why?
Cortisol levels can be measured using various collection methods, depending on the clinical context and patient characteristics.
The table below compares the three main testing methods:
| Test Type | Key Characteristics | Common Indications | Key Advantages |
| Serum cortisol | Blood sample, typically collected between 07:00–09:00 | General assessment of adrenal function | Standard method, widely available |
| Salivary cortisol | Non-invasive, at-home collection; ideal for night-time levels | Diagnosis of Cushing’s, circadian rhythm analysis | Reflects free cortisol, ideal for chronic stress |
| 24-hour urinary cortisol | Urine collected over a full 24-hour period | Evaluation of chronic cortisol overproduction | Provides an average of daily secretion |
4. Why choose salivary cortisol testing?
El uso del cortisol salival se ha expandido, proporcionando un diagnóstico más preciso y accesible, especialmente en contextos clínicos e investigaciones sobre el estrés y disfunciones del eje hipotálamo-hipofisario-adrenal (HHA).
Salivary cortisol testing has gained prominence as an accurate and accessible method, especially in clinical contexts and research involving stress and HPA axis dysfunction.
Scientific evidence shows that salivary testing accurately reflects the biologically active, free fraction of cortisol. It is particularly useful in evaluating chronic stress, cognitive alterations, anxiety, depression, panic disorder, sleep deprivation, and chronic fatigue.
In hormonal disorders, especially at night, salivary testing has shown superiority over plasma cortisol for diagnosing Cushing’s syndrome (CS), particularly in cases of mild or intermittent hypercortisolism.
Recent studies suggest that midnight salivary cortisol has greater diagnostic sensitivity.
Moreover, comparison studies indicate that the dexamethasone suppression test (DST) demonstrates improved diagnostic precision when performed using salivary samples. Healthcare professionals should be aware of variations between laboratory assays and ensure interpretation is based on validated standards.
5. Preparation for different types of cortisol testing
- Serum cortisol: Fasting for at least 4 hours is recommended, and the patient should inform their doctor of any medications that may interfere with the results (e.g. corticosteroids, contraceptives, antidepressants).
- Salivary cortisol: Avoid eating, drinking, or brushing teeth for at least 30 minutes before collecting the saliva sample.
- Urinary cortisol: Collect all urine produced over 24 hours in a container provided by the laboratory.
6. Interpreting cortisol test results
- Elevated levels: May indicate Cushing’s syndrome, ACTH overproduction by the pituitary gland, cortisol-secreting adrenal tumours, prolonged corticosteroid use, or chronic stress.
- Low levels: May suggest adrenal insufficiency, Addison’s disease, secondary adrenal insufficiency, or hypopituitarism.
Interpretation should always be carried out by a qualified healthcare professional, considering the clinical context, time of sample collection, and any medications that could affect cortisol levels.
7. The relevance of cortisol in mental health
Research indicates that elevated cortisol is frequently associated with chronic stress, contributing to mental health disorders such as anxiety, depression, and sleep disturbances.
Professionals exposed to high-stress environments — such as medical staff or educators — may experience persistent high cortisol levels, negatively affecting both mental and physical health.
On the other hand, chronically low cortisol levels — as in adrenal insufficiency — can result in psychological symptoms such as apathy and low motivation.
In such cases, cortisol testing combined with other evaluations may provide critical insight for accurate diagnosis and treatment.
8. Treating cortisol-related disorders
- Cortisol excess: Treatment may include medication to inhibit cortisol production or, in severe cases, surgical removal of ACTH- or cortisol-producing tumours.
- Cortisol deficiency: Commonly treated with hormone replacement using synthetic corticosteroids.
In addition, Non-invasive NESA Neuromodulation presents a promising approach to regulating the HPA axis and modulating autonomic nervous system dysfunctions.
This technique influences neural activity in regions such as the prefrontal cortex and amygdala.
Recent studies indicate that this intervention can help reduce hypercortisolism in patients with chronic stress, anxiety, and depression, as well as optimise physiological responses in individuals with cortisol deficiency.
NESA WORLD recommendation
While cortisol tests provide valuable information, results must be interpreted by a specialist physician, considering the full clinical context and, when necessary, complementary examinations.
In scientific research, salivary cortisol testing stands out as a non-invasive and effective alternative. It allows sample collection at different times throughout the day, minimising the influence of external variables.
For studies on chronic stress, sleep disorders, and psychological health, this testing method provides precise data and is essential for developing new therapeutic strategies.
Conclussion
Cortisol testing is an indispensable diagnostic tool for assessing endocrine health and identifying stress-related or adrenal disorders.
Understanding cortisol levels provides a comprehensive view of hormonal balance and its implications for both physical and mental health. Accurate result interpretation and a personalised therapeutic approach are essential for effectively managing cortisol-related endocrine and psychological conditions.
Reference
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2. Paganin, A., & Signorini, S. (2024). Salivary cortisol in schizophrenia: A selective review and meta-analysis of controlled studies of the past decade. Biomarkers in Neuropsychiatry, 11, 100098. https://doi.org/10.1016/j.bionps.2024.100098
3Smeets, M. M., Vandenbossche, P., Duijst, W. L., Mook, W. N. V., & Leers, M. P. G. (2021). Validation of a new method for saliva cortisol testing to assess stress in first responders. Emergency Medicine Journal, 38(4), 297–302. https://doi.org/10.1136/emermed-2019-209205
4. Giacomello, G., Scholten, A., & Parr, M. K. (2020). Current methods for stress marker detection in saliva. Journal of Pharmaceutical and Biomedical Analysis, 191, 113604. https://doi.org/10.1016/j.jpba.2020.113604