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People often ask whether they should get their endogenous melatonin levels assessed. There are some considerations to be aware of before testing through a qualified health professional.




Melatonin is found in most body fluids, including blood, saliva, and urine (1), therefore, testing options are available for each of these body compartments. Note that what is of interest is assessing the melatonin produced by the pineal gland, which responds to darkness; therefore, the light-dark conditions must be considered in all cases.(2,3) Exposure to <30 lux is recommended, though some researchers have reported <10 lux.(2)


Further, the testing options are most commonly used to determine dim-light melatonin onset (DLMO), which is the approximate time for the synthesis and secretion of melatonin, typically 2-3 hours prior to routine sleep.(2)  


Due to the circadian rhythm of melatonin, samples should be collected in the evening to early morning hours, and more frequent samples improve accuracy. (3) Further, each method provides a different measurement, and therefore, one test result should not be compared to another. For example, plasma melatonin is about three times greater than that collected from saliva; therefore, 3 pg/mL of saliva would be comparable to approximately 10 pg/mL of plasma. (2)


Testing options


Saliva: Salivary testing is considered a reliable, non-invasive method to measure melatonin at the time of collection. It can be used to determine dim-light melatonin onset (DLMO), a parameter that can be used to diagnose and monitor circadian rhythm disorders, therefore, it is collected in the evening hours. (3,4) About 30-40% of the melatonin in the plasma is excreted into saliva through passive diffusion. It is reported in some studies that salivary testing is a comparable marker to serum testing. (3,4) However, other studies have not confirmed this connection. (5) 


Since pineal melatonin is so strongly suppressed by light exposure (6), an individual must be in dim light conditions (<30 lux) for four to seven hours in the evening during saliva sample collection to accurately determine when melatonin starts to rise.2 Collection times have been reported as a 6-hour testing window (5 hours before scheduled bedtime and 1 hour after scheduled bedtime), up to a 20-hour testing window in either 30-minute or 60-minute collection times. (2,4) However, Crowley et al., reports that along with other studies, their current analysis shows that collections every 60 minutes for a 6-hour window are as accurate as the collection every 30 minutes and more feasible in a clinical setting than collection times up to 20 hours per day. (4)


Urine: Melatonin, as 6-sulfatoxymelatonin (aMT6s) is the main metabolite excreted in the urine, though a small amount (about 1%) of unmetabolized melatonin can also be found in the urine, which can also be measured.3 It has been reported that aMT6s is a good comparison marker or projection of plasma melatonin levels due to the correlation between these two tests.(3,5,7,8) It is worth noting that when taking oral melatonin, it is collected in the blood, but 30-60% is immediately metabolized in the liver to aMT6s and excreted in the urine. Therefore, urinary aMT6s can increase after supplementation, but this measure would not then be reflective of melatonin levels in the blood. (7)


Urinary melatonin assessment is more easily achieved with a single sample of first-morning urine, though more frequent collections, every 2-8 hours for 24-48 hours, help to ensure accuracy.(2,3) This measure can provide an assessment of the total nocturnal melatonin release. (3) The results can be influenced by how melatonin is metabolized in the liver and overall kidney function.(3)


Blood: Melatonin made in the pineal gland is distributed to the body through the blood, which is then metabolized through the liver and excreted as aMT6s in the urine.7 It is important to recognize that melatonin has a short half-life, between 20-60 minutes, in the blood. (3) Further, blood samples only reflect the amount of melatonin circulating at the time of collection. Due to this and the short half-life, frequent sampling, about every 20-30 minutes, is needed for an accurate assessment of circadian rhythm.(3,5) As a result, an intravenous catheter is used, making it impractical in most clinical settings. (2)


Peak plasma concentrations are achieved between 3-4 am, with the lowest from 7-9 am. It is expected that levels throughout the day would be low or undetectable. (3) It is estimated that daytime plasma melatonin levels are maintained at approximately 5 pg/mL, whereas nighttime levels can range from 50-100 pg/mL. (3) Blood samples are a sensitive measure that can give a real-time assessment of melatonin levels and can be useful in incidences of low melatonin that could not be easily measured in saliva or urine.  

Considerations prior to testing


While this list is not exhaustive, these are some considerations to account for prior to testing (3):

  • Changes in posture, particularly standing, can increase the plasma concentration of melatonin, though it can be reversed within 10 minutes. Salivary melatonin secretion has also been noted to increase in standing over supine positions.(9) While there is not an ideal postural position for collection, minimizing postural changes and movement prior to plasma and saliva testing is recommended.(2)

  • Disturbed sleep can provide false results in melatonin levels; therefore, the person should be provided with an environment that promotes restful sleep.

  • Age is a factor in the amount of melatonin produced, with the highest levels in childhood and the lowest levels in adults ages 50 years and older. 

  • Genetics may alter melatonin secretion. A recent, first conducted genome-wide association study (GWAS) found five genetic variants in a Taiwan Han Chinese population that influence melatonin secretion.(10)

    • ZFHX3 (rs17681554)- further studies are needed to understand the linkage to melatonin

    • GALNT15 (rs142037747)- it remains to be determined how and if this significantly affects melatonin levels

    • GALNT13 (rs7571016)- it remains to be determined how and if this significantly affects melatonin levels

    • LDLRAD3 (rs9645614)- a possible mechanism for altered melatonin levels in individuals with Alzheimer’s disease

    • SEPP1-FLJ32255 (rs6451653)-protects the plasma membrane from oxidative damage

  • Gender may alter melatonin secretion. For example, it is reported that levels of aMT6s in women (but not men) are lower with higher BMI and smoking status. Serum melatonin levels are lower in men who are current smokers but slightly higher in those who are obese, whereas obese women have lower levels of melatonin. (8)

  • Nocturnal melatonin release is influenced by the ubiquitous electric light in our homes in the evening. Light exposure influences melatonin secretion by the pineal gland. Avoidance of blue lights (i.e., electronics) is required for accurate testing. Exposure of less than 30 lux or the use of red light is recommended.

  • Physical activity at night time can delay or suppress melatonin production.

  • Melatonin secretions have seasonal variations, with more melatonin secretion in the winter months compared to the summer months, though this was not statistically different. (8)

  • Multiple health conditions and/or organ dysfunction can alter melatonin secretion, metabolism, and/or production and therefore impair test results. Conditions to note include ophthalmic disease, spinal cord injuries, liver and kidney diseases, and periodontal diseases.  

  • Some medications may impact melatonin levels, either increasing or decreasing production. This list includes, but is not limited to, hormonal birth control, some benzodiazepines, beta-blockers, NSAIDs, GABAergic drugs, MOA inhibitors, some SSRIs, and antiepileptic drugs.

  • The use of melatonin supplements can interfere with determining the amount of melatonin produced by the body, therefore, discontinuing supplementation prior to the test may be recommended. 

  • Some labs may require the avoidance of certain foods before testing.

  • Elevated cortisol levels at night will decrease melatonin production, therefore, testing cortisol levels may also benefit some individuals.


With multiple influencing factors and considerations to account for, the value of testing melatonin levels for clinical practice has not been established. Urine testing may be the ideal choice for testing since it has been reported to reflect blood levels, requires only one collection, it is easy to collect, non-invasive, it is not impacted by exposure to light, and can be easily stored for processing.(7)

Summary of testing options

Measurement of
Melatonin circulating at the time of collection (5)
Morning aMT6s measure peak nocturnal melatonin levels (8)
Melatonin circulating at time of collection (8)
Immediately suppressed by light
Easily collected
Collected at home
Placed in freezer for delivery to the labs
Can be stored at room temperature for up to 5 days or 20 degrees C for 2 years (5)
Proper handling and storage of the sample is required by the lab.(3)
Collection considerations
Do not brush teeth or consume liquids 30 minutes prior to testing. Avoid lipstick or other lip products. Use of cotton swabs or paraffin chewing to collect saliva.
First pass urine collection. For frequent sampling, a catheter may be used.
Often requires catheter for frequent sampling and therefore not recommended for routine clinical use. (2)
Requires low light (<30 lux) prior to collection
Number of samples
Every 30-60 minutes, generally for 6 hours.
Once, in the morning or every 2-8 hours for 24-48 hours for increased accuracy.
Used to determine
Total nocturnal melatonin release
DLMO Cases of low melatonin (as this may not be captured in urine or saliva)

Written by Catherine Darley, ND & Kim Ross, DCN

Reviewed by Deanna Minich, PhD

December 5, 2022




1.        Tan DX, Xu B, Zhou X, Reiter RJ. Pineal calcification, melatonin production, aging, associated health consequences and rejuvenation of the pineal gland. Molecules. 2018;23(2). doi:10.3390/molecules23020301

2.        Benloucif S, Burgess HJ, Klerman EB, et al. Measuring melatonin in humans. Journal of Clinical Sleep Medicine. 2008;4(1). doi:10.5664/jcsm.27083

3.        Rzepka-Migut B, Paprocka J. Melatonin-Measurement Methods and the Factors Modifying the Results. A Systematic Review of the Literature. Int J Environ Res Public Health. 2020;17(6):1916. doi:10.3390/ijerph17061916

4.        Crowley SJ, Suh C, Molina TA, Fogg LF, Sharkey KM, Carskadon MA. Estimating the dim light melatonin onset of adolescents within a 6-h sampling window: The impact of sampling rate and threshold method. Sleep Med. 2016;20. doi:10.1016/j.sleep.2015.11.019

5.        Mirick DK, Davis S. Melatonin as a biomarker of circadian dysregulation. Cancer Epidemiology Biomarkers and Prevention. 2008;17(12). doi:10.1158/1055-9965.EPI-08-0605

6.        Kubota T, Uchiyama M, Suzuki H, et al. Effects of nocturnal bright light on saliva melatonin, core body temperature and sleep propensity rhythms in human subjects. Neurosci Res. 2002;42(2). doi:10.1016/S0168-0102(01)00310-8

7.        Kovács J, Brodner W, Kirchlechner V, Arif T, Waldhauser F. Measurement of Urinary Melatonin: A Useful Tool for Monitoring Serum Melatonin after Its Oral Administration. J Clin Endocrinol Metab. 2000;85(2). doi:10.1210/jcem.85.2.6349

8.        Nogueira LM, Sampson JN, Chu LW, et al. Individual variations in serum melatonin levels through time: Implications for epidemiologic studies. PLoS One. 2013;8(12). doi:10.1371/journal.pone.0083208

9.        Kozaki T, Arata T, Kubokawa A. Salivary Melatonin Concentrations in a Sitting and a Standing Position. Journal of Hormones. 2013;2013. doi:10.1155/2013/236325

10.      Liu PH, Chuang GT, Hsiung CN, et al. A genome-wide association study for melatonin secretion. Sci Rep. 2022;12(1). doi:10.1038/s41598-022-12084-w




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