Saliva diagnostics: The basics

It has been over four decades since the use of a saliva sample for steroid analysis was first documented in scientific literature.1

Image Credit: Tecan

Since then, saliva sampling and analysis have developed into an appealing and beneficial alternative to blood testing due to its easily repeatable, non-invasive nature.

Not only is it less stressful and more convenient for patients, but saliva sampling also allows for the collection of multiple samples and is a viable technique in settings where blood sampling may not be safe or practical. 2

Saliva-based diagnostics offer the potential to revolutionize the personalized medicine field due to their capacity to assess a wide range of physiological conditions from stress and depression to sex hormone imbalances.

Saliva’s usefulness stems from its inclusion of a number of chemicals and hormones which serve as either direct indicators or indirect biomarkers of biochemical imbalances, certain disease states and overall human health.3

The use of saliva testing has extended beyond simple sex hormone testing in recent years, moving into much wider fields, including sleep disorders, occupational and sports medicine.4,5

Benefits of saliva testing

A number of saliva-based diagnostic assays are currently available.

These assays can be used to detect and measure a range of medically relevant compounds, including steroids, the adrenal hormones dehydroepiandrosterone (DHEA) and cortisol, the sex hormones progesterone, estradiol and testosterone, and substances such as IgA, melatonin and alpha amylase.

Each of these tests has distinct advantages when performed via saliva testing. They can be performed in a non-invasive, easy, painless and rapid manner and are practical as they can be done anywhere at any time.

The ease and safety afforded by saliva testing methods mean this can even be done at home by a patient or lay person, ideal for circumstances or conditions requiring testing or analysis in remote locations or outside of normal working hours.

Another key benefit of saliva measurements is that they can reflect biologically active steroid levels.6

Almost all steroid hormones in the bloodstream are effectively bound to carrier proteins and are therefore metabolically inactive. Only a small amount of any blood-borne hormone is left unbound and able to enter cells and affect their function.7

Due to this, standard blood tests are not a viable means of measuring bioavailable hormone levels.

These free, unbound hormones will exit the bloodstream via the membranes before entering saliva. Therefore, saliva testing is a robust means of measuring biologically active hormones.

Established approaches to saliva testing have also proven to be extremely accurate.6 Saliva contains far lower concentrations of hormones that are present in blood (just 1-5% of total hormone levels), meaning that it is vital that saliva-based assays are sensitive enough to measure these low levels (Figure 1).

Steroids in blood and saliva: the case for saliva testing

Figure 1. Steroids in blood and saliva: the case for saliva testing. Image Credit: Tecan

Barriers to market acceptance

Overall, the market acceptance of clinical saliva testing has been hindered by a combination of limited awareness of its benefits in the clinical community and the limited number of accepted SOPs available. Established SOPs used in clinical settings still tend to be blood-based for these reasons.

In contrast, saliva-based testing is widely used and well established in the ‘wellness’ clinical setting, despite the number of labs making use of saliva for hormonal testing being comparatively small in the established medical sector.

Wellness practitioners are typically happy to perform saliva testing due to its ease of use and accessibility. These practitioners cannot prescribe, however. There is a mismatch between this level of adoption and that of physicians, who can prescribe but tend to use blood testing.

There are a number of reasons for this dichotomy; for example, ‘wellness’ is a common 21st-century goal, with thousands of online tests – including DNA and hormone tests – at the forefront of this emerging industry.8

Unfortunately, some of these tests suffer from an exceeding lack of scientific foundation, and there is a notable lack of standardization across the sector.9

Female hormone testing: From managing infertility to menopause

A key application area where saliva testing has become routine is in the measurement and evaluation of female sex hormones.

Blood tests have been the primary means of measuring female hormone levels for decades, but blood sampling is expensive, invasive and frequently logistically challenging.10

These difficulties have prompted a shift towards the testing of more convenient and cost-efficient sample types, including saliva.

Saliva-based tests have been proven to be an accurate, reliable means of measuring female hormone levels as well as being painless for the patient.

Hormone levels change with age (Figure 2), and measurement of these and other related hormones can afford medical professionals valuable insight into any hormone imbalances, enabling them to recommend an appropriate course of treatment.

Variation in sex hormone levels with ageFigure 2. Variation in sex hormone levels with age. Image Credit: Tecan

Symptoms of a potential hormone imbalance may include excessive menstrual bleeding, infertility or hot flashes in menopause. Once a medical practitioner has identified these symptoms, they will first look at measuring the level of specific hormones.

Blood samples would traditionally be taken, and serum-based tests would be performed, but saliva-based hormone testing has provided a less invasive and more cost-effective means of measuring female hormones.

A saliva test is comprised of three parts: sample collection, hormone measurement and downstream analysis.

Sample collection has historically been a significant source of variation, negatively impacting the uptake of saliva-based testing. SOPs are now in place, however, detailing appropriate sample collection methods and collection devices, as well as an array of recommended operating procedures for saliva testing labs.

Recommended platforms for measuring and analyzing most hormone levels in saliva samples are either enzyme immunoassay (EIA, for example, ELISA) or mass spectrometry-based (i.e., lc-MS/MS).

Almost any start-up lab can perform ELISA tests effectively. These can be performed manually or using automated workstations. Centralized service labs working with very high volumes of samples may offer more expensive lc-MS/MS options.

Once samples have been analyzed and hormone levels have been evaluated against normal ranges, results can be shared with the clinician. The clinician can then take action and prescribe treatment as appropriate, including hormone replacement therapy (HRT).


  1. Landman, A. D., Sanford, l. M., Howland, B. E., Dawes, c., & Pritchard, E. T. (1976). Testosterone in human saliva. Experientia, 32(7), 940–941. PubMed ID: DOI:
  2. Pujari, M., Bahirwani, S., Balaji, P., Kaul, r., Shah, B., Daryani, D., & Iqbal, S. (2012). Oral fluid nanosensor test: saliva as a diagnostic tool for oral health. Journal of the california Dental Association, 40(9), 733–736. PubMed ID :
  3. Bellagambi, F.g., lomonaco, T., Salvo, P., vivaldi F., Hangouet, M., ghimenti, S., Biagini, D., Di Francesco, F. & Fuoco, r. & Errachid, A. (2020). Saliva sampling: Methods and devices. An overview. Trends in Analytical chemistry. Elsevier. volume 124, March 2020, 115781 Elsevier ID: DOI:
  4. Rahman, S. A., Kayumov, l., Tchmoutina, E. A., & Shapiro, c. M. (2009). clinical efficacy of dim light melatonin onset testing in diagnosing delayed sleep phase syndrome. Sleep medicine, 10(5), 549–555. PubMed ID: DOI:
  5. Chojnowska, S., Ptaszyńska-Sarosiek, I., Kępka, A., Knaś, M., & Waszkiewicz, N. (2021). Salivary Biomarkers of Stress, Anxiety and Depression. Journal of clinical medicine, 10(3), 517. PubMed ID: DOI: 10.1016/j.sleep.2008.03.020
  6. Lewis J. g. (2006). Steroid analysis in saliva: an overview. The clinical biochemist. reviews, 27(3), 139–146. PubMed ID:
  7. Accessed 29 June 2021
  8. Accessed 10 August 2021.
  9. Dr Gonshor, personal observations.
  10. Sufi, S. B., Donaldson, A., gandy, S. c., Jeffcoate, S. l., chearskul, S., goh, H., Hazra, D., romero, c., & Wang, H. Z. (1985). Multicenter evaluation of assays for estradiol and progesterone in saliva. clinical chemistry, 31(1), 101–103. PubMed ID: DOI:


Produced from materials originally authored by Dr. Aron Gonshor from FLUIDS iQ.

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Last updated: Jan 31, 2024 at 9:19 AM


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