Testing for Steroid Hormones

Testing for steroid hormones. Most practitioners wish to get an idea of their patient’s steroid hormone status. They would further like to confirm that there is a positive result which can be demonstrate by rechecking testing.  Commercial labs are available to provide measurement of hormones in blood, saliva, urine and even hair and fingernails.  To provide reasonable information, lab tests should provide great correlation with the clinical signs of deficiencies or excesses.

The different types of hormone tests vary in their sampling and analysis techniques. Each has its own proponents and critics, as well as limitations. Before beginning hormone testing, determine which type of test is most appropriate for your needs.  These lab tests are designed for hormones like sex and adrenal hormones derived from cholesterol.

Urine Tests

container for urinalysis in the hand of the doctor

The oldest method of testing for steroid hormones is urine testing. It is relatively easy to measure hormones in urine because larger amounts of hormone appear in urine than in blood or saliva. Urine testing requires that the patient collect every drop of urine for 24 hours.

Hormone identification in urine is usually achieved using gas chromatography to separate the hormones and then identifying the hormone with mass spectroscopy.  Although this method does not identify the high and low levels that can occur within normal daily fluctuations, it is still a good measure of how much of a specific hormone is available on that day.  Urine tests also track specific hormones plus their metabolites, which helps assess more information. This technique offers excellent separation efficiency and high sensitivity, making it suitable for even low-concentration hormone measurements.

The test is not accurate if only some of the urine is collected over the 24-hour period, if some of the urine is discarded, or if kidney function is impaired. Urine tests are standardized with creatinine to ensure kidney function is normal.

A variation on 24-hour urine collection measures hormones in urine samples collected at timed intervals throughout the day. There is an assumption that timed samples can reflect the same data as collecting all the urine.  Current labs use liquid chromatography with mass spectrometry (LC/MS) which has high hormone specificity.

Blood Tests

A method of blood testing for steroid hormones is serum testing, in which one or more vials of a patient’s blood are collected during a laboratory appointment and the separated serum is measured for hormone levels. Serum measurements account for hormones bound to carrier proteins and small amounts of unbound hormones.  Most labs now process the serum samples using LC/MS. 


Immunoassays have been used in testing for steroid hormones due to the convenience, speed, and cost-effectiveness. These assays utilize the specific binding between an antibody and the target hormone to quantify its concentration. The most common immunoassay formats used for steroidal hormones include enzyme-linked immunosorbent assays (ELISA) and radioimmunoassay (RIA). ELISA relies on the detection of an enzyme-linked signal, while RIA employs radioactive tracers for measurement. Although immunoassays are widely accessible and provide rapid results, they may exhibit cross-reactivity with structurally similar hormones, potentially leading to inaccurate measurements.  This method had been commonly used for serum and continues to be used for saliva analysis.

Issues with Serum Testing

Critics of serum testing claim that the invasive procedure itself can alter the circulating hormones. They also contend that serum measurements do not account for the hormones carried by the red blood cells, which are separated out from the serum, and which may have a higher hormone concentration inside the cell. Others counter this argument with the explanation that, even though small amounts of hormones are found in the serum, it is only the hormones in the serum that are available to other tissues in the body, so the measurement is accurate.

Another downside with this collection method is that, unlike a urine test, this type of blood sample is just a snapshot in time. The levels of the various hormones in the bloodstream can change from moment to moment and from day to day. For instance, if a perimenopausal woman’s blood sample indicates that her estradiol level is in the normal range, it does not necessarily mean that she has a normal amount all day long; she could be higher or lower at any given time throughout the day or even throughout the month.  Correlation to the amount of hormone in tissues is weak. This suggests that testing for steroid hormones with serum methods may not be entirely reliable.

Blood Spot Testing

Another form of blood collection called blood spot testing can account for these hormone fluctuations. It can be scheduled for specific days of the month (such as before or after hormone therapies), or specific times of the day (such as before or after meals or other medications). For blood spot testing, a patient collects drops of their own blood from a finger prick onto a card for submission to a lab for analysis. With this method, results are typically comparable to those from serum tests, but without the cost and inconvenience of a lab visit. Sample collection can be affected by milking the finger for more blood and thus including interstitial fluid with the capillary blood.  Small sample sizes can limit the number of hormones that can be tested.

When using serum testing to monitor treatment, it is important to understand the dosage forms used and the timing of the blood sample collection.  If using hormones in implanted pellets, injectables or patches, the blood levels are fairly consistent so can be tested anytime.  Practitioners can decide if they have to test at the peak time of these dosage forms or as the blood levels will be waning.  In short acting dosage forms, such as capsules, tablets, creams, gels, sublingual such as troches, vaginal or rectal suppositories, peak levels will occur in 1 to 2 hours, midlevel’s in 4 to 6 hours and almost gone from the blood stream in 12 hours.  It is very important to be consistent each time blood samples are taken.

Saliva Tests

Saliva testing is also commonly used to evaluate hormone levels Immunoassays are used to analyze hormones from a collection of saliva. Similar to blood tests, each saliva sample represents only a snapshot.

Saliva samples are non-invasive and easy to collect with the necessary frequency. Some commercial labs offer saliva test kits for collecting several samples throughout a day or a month, so that they have more data to evaluate. One potential drawback is that saliva samples can be easily contaminated if disease is present, or if blood gets into the saliva sample.  Some bacteria found in the mouth microbiome can produce steroid hormones.  Lab instructions require no eating, coffee, brushing teeth and chewing gum 30 minutes before collecting the saliva samples.

Saliva tests tend to be more accurate with younger people than with older individuals because, in general, they have higher levels of hormones available. When measuring the hormones of a menopausal woman, for example, saliva tests are at their limits of sensitivity. This is since the amount of hormone available for measurement is so small that test results may be inconsistent or inaccurate.


Because there are so many steroidal hormones, and because their chemical structures are similar to each other, there might be “spill over” from one hormone to other similar hormones when using the immunoassay methods.

Proponents of saliva tests claim that only the “free” hormone is filtered from the blood into the saliva. Therefore, these tests provide a more accurate measure of the hormone that is biologically active or available to other tissues. However, there is still some difficulty in evaluating the results of hormone supplements with some dosage formulations because the impact is far more dramatic in saliva than it is in blood or urine.  The bacteria in the mouth microbiome are also able to produce steroid hormones.

Hair and Fingernails

Like other body tissue, hormones are present in the hair and fingernails and may be measured.  These measure hormone levels over longer periods of time and changes in hormone therapy may be only reflected over longer periods of time. Reference ranges are still being developed.

How Reliable Are Hormone Tests?

A hormone test provides a single measure of one or more hormones, which your healthcare practitioner will compare against a prior measure and/or against the broad ranges of “normal” hormone levels that have been accepted as the standard criteria. However, there are significant variations across populations, genders, ages, and even within individuals, that must be factored into the equation. For instance, women have higher levels of estrogens and progesterone than men, and women who are still menstruating have higher levels than menopausal women.

In the case of testosterone, more of the available testosterone becomes tightly bound to sex hormone-binding globulin (a water-soluble carrier protein), which increases with age. To account for this, some practitioners will also order a test to measure sex hormone-binding globulin to get a better sense of testosterone activity. Men generally have higher DHEA and testosterone levels than women and, in both sexes, the available levels decrease with age.

Bioidentical Hormones

The hormone tests that are currently available only measure “bioidentical” hormones: that is, only hormones which are chemically identical to those that exist naturally in our bodies. However, some hormone replacement therapies include animal-based hormones and synthetic analogues of hormones. These are not measured by hormone tests but probably affect the balance of other hormones being measured. Synthetics can block the production of the body’s hormones. Synthetics are not measured when testing for steroid hormones.


Hormone tests also do not measure what is happening at receptor sites. Receptor sites are the final step to actual hormone activity. For instance, birth control hormones may affect hormone measurements because they occupy receptor sites, with the result that the body may not perceive a need for hormone production.  Receptors are promiscuous. Xenoestrogens and heavy metals, for example, can bind at estrogen receptors.

Nor do hormone tests evaluate tissue concentration. For example, estrogens concentrate in tissues such as the uterus. The only technique known for evaluating this tissue would be to surgically remove bits of the tissue and then extract and measure the hormone found in that tissue. 

Testing for steroid hormones cannot predict the best dosage form or dose for an individual.  Testing must be coherent with signs and symptoms to be of value.

Unlike previously assumed, steroid hormones are not only produced by specific organs such as the ovaries, testes and adrenal glands.  Hormones are made independently in the Schwann cells in the nervous system tissue, inside mast cells and other cells (known as intracrinology) and in the brain.  This hormone production may never show up in the body fluids used for testing.

When Should You Get Tested for Steroid Hormones?

A comprehensive understanding of your total hormone picture—that is, the balance, —may be more important than the exact measure of a few specific hormones. To optimize testing, check as many hormones as possible.   A hormone test, which represents a single measure (or a single day’s measure) of one or more hormones, can be a limited tool.  Testing should be combined with clinical observation and monitoring of symptoms to help determine a hormone deficiency or excess, and if hormone therapy may be an appropriate.

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