Low Testosterone Diagnosis: Blood, Urine and Saliva Tests

The number of men diagnosed with low testosterone has grown dramatically in recent years.

If you have been experiencing low energy, mental fog, less interest in sex, and poor mental performance, all of this can be due to a fall in your testosterone levels.

If low T remains untreated, it can lead to stunted reproductive growth, weak muscle, porous bones, and mood disorders. However, such symptoms are not always highly specific to low testosterone.

For some people, these symptoms may be due to other medical problems. For others, low testosterone may go undetected until more complications occur.

Identifying Causes of Low Testosterone

Identifying the cause of low testosterone is vital for deciding on the course of treatment to correct the problem.

Testosterone levels normally decline as we age. Testosterone production spikes up during puberty. It stays relatively high in your 20s. But once you hit your 30s, it starts to dip. As we grow older, into our 40s, 50s, and 60s, our testosterone levels tend to fall significantly. But due to other causes, testosterone levels fall below the minimum requirements.

Low T is diagnosed when testosterone levels fall below 300 ng/dL (x). It’s best to maintain at a level more than 600 for optimal health. Any irregularity in the production of testosterone can cause abnormally low T. 

Read more: The causes of low testosterone

Low T can be diagnosed accurately with the help of your doctor. They will ask about your symptoms and medical history. They will order tests to identify the cause of your low T. The doctors will be able to guide you on the right course of treatment after that.

Investigation: Diagnosing Low Testosterone

Blood tests, urine tests, saliva tests, and imaging tests are used for diagnosing testosterone deficiency. 

Blood Tests for Diagnosing Low Testosterone

Before getting to the types of tests, you need to be aware that testosterone is present in our body in two forms: Free testosterone and Protein-bound testosterone. 

  • Free testosterone: Only 2 to 3 percent of the testosterone is free (x). They are considered bioactive. Free testosterone can interact with the tissues and cells in your body by connecting to cell receptors. From 20-25 years, the free testosterone levels should ideally be 5 to 25 ng/dL. For 25-30 years, the levels are 5.05 to 19.8 ng/dL (1).
  • The bound testosterone: About 97 percent of the testosterone in the body is in the bound form. These testosterones are bound to protein. They may be bound to either Albumin or SHBG (sex-hormone binding globulin). The testosterone is loosely bound to albumin, is bioavailable, and can be easily used by the body. However, the testosterone bound to SHBG is unavailable (2). A high level of SHBG level reduces the effects of testosterone by binding a greater amount of testosterone in the blood, slowing down testosterone’s uptake by the cells.  

Now let’s look at the tests conducted with regard to these forms of testosterone:

Total Testosterone Levels

The normal range of total testosterone is 300-1000ng/dL. This includes both free testosterone and protein-bound testosterone. If the test result is lower than 300mg/dL, it is considered as low T.

The optimal condition for giving the blood sample for the test:

  1. Be sedentary 24 hrs before – avoid intense exercise, emotional stress, sexual intercourse as all these activities overconsume testosterone and transiently reduce testosterone levels.
  2. Drink 3 glasses of water to avoid dehydration.
  3. Empty the stomach to avoid food interferences.

As the testosterone levels are at their highest in the morning, this test is usually performed between 7 am to 10 am. This test may need to be done more than once to get an accurate value.

Testing Free Testosterone Levels

If the total testosterone in the blood detects low T, then free testosterone level needs to be checked.

SHBG Testing

The SHBG (Sex Hormone-Binding Globulin) is produced in the liver. SHBG binds to testosterone and delivers it to the tissues for use. An SHBG test is often done to check the amount of testosterone that is going to the tissues.

A low SHBG result shows that more testosterone is unattached and free in the system. This means there is too much testosterone available in the body. A high SHBG test shows that less testosterone is available to the body to use. This particular test helps identify low testosterone in men. SHBG tests also help identify excess testosterone in women especially the ones with PCOS (3).

Blood Tests for Identifying Cause of Low Testosterone

Low testosterone in men is also known as “hypogonadism” although this term is often used in reference to reduced production of all sex hormones, not just testosterone.

Once the above-mentioned blood test confirms low T, there are other tests that can help categorize the type of hypogonadism. They are of two kinds:

Primary hypogonadism: The testis in males and ovaries in females are known as gonads. If the low testosterone occurs due to a problem with the gonads, the condition is called primary hypogonadism (4).

Secondary hypogonadism: Secondary hypogonadism is diagnosed if the cause of low testosterone is a problem with the pituitary gland or the hypothalamus. These are the two organs that send messages to the gonads to secrete testosterone (5).

Testing FSH, LH, Prolactin, Estradiol, and Estrone levels help detect whether you have primary or secondary hypogonadism.

FSH Test

The Follicle-Stimulating Hormone (FSH) is produced by the pituitary gland. In men, this hormone helps in the production of sperm and estradiol production in the testicles. FSH is also important in producing androgen-binding proteins (6). FSH test can help identify if the low testosterone is due to a pituitary disorder. 

In women, FSH controls the menstrual cycle and the formation of eggs. Too much or too little FSH can cause many problems such as irregularities in the menstrual cycle, delayed puberty, and even infertility (7).


  • deficient – 0 to 0.9
  • optimal – 2
  • excess – >5

LH Test

The Luteinizing hormone (LH) is another important hormone produced by the pituitary. It is very important for both men and women, for their sexual development and fertility. LH stimulates testosterone production by the Leydig cells in the testicles.

Measuring the amount of LH in the blood can help diagnose if the underlying cause of your low testosterone is a pituitary disorder.

Serum LH levels:

  • deficient level – <2 mIU/ml
  • optimal level – 2 to 3 mIU/ml
  • excessive level – >6 mIU/ml

Prolactin Test

Prolactin is a hormone produced by the pituitary gland. In men, prolactin helps in testosterone secretion that helps in sperm production. In females, it helps in breast development and the production of milk. But high prolactin leads to hypogonadism leading to less T production.

Prolactin test is done to investigate testicular dysfunction in men. It can also help point out whether the problem is in the pituitary gland or hypothalamus. High levels of prolactin can be a marker of pituitary tumors, in both men and women (8).

Serum prolactin levels

  • deficient – 0 – 80mIU/l
  • optimal – 100 – 300mIU/l
  • excessive – > 400 mIU/l

Estrogen Test

Estrogen is the primary sex hormone in females. Men also produce estrogen but in lesser quantities. Estrogen is actually an umbrella term for the three hormones Estradiol (E1), Estriol (E2), and Estrone (E3).

In men, some of the testosterone is converted into estrogen. Along with testosterone, estrogen helps regulate sexual development. Estrogen is needed for boosting sex drive, sperm production, and maintaining an erection (9). Low T can result when estrogen levels are too much or too little.

When estrogen levels in men go too high, the body downregulates the production of testosterone to prevent more estrogen creation. So high estrogen levels in men can cause low T.

Insulin resistance and fatty liver can also cause excess estrogen. The liver is the main site where estrogen metabolism takes place. Estrogens are metabolized and inactivated in the liver. This is a process by which your liver gets rid of the excess estrogen. Therefore, if your liver is inflamed or damaged, it impairs this functioning resulting in an increase in estrogen levels in your blood. If this condition coincides with low T, the hypogonadism is much more aggravated (10).

Many of the symptoms of low T are actually caused by estrogen deficiency in men (11). Estrogen testing helps identify whether the low testosterone is due to low estrogen levels or estrogen excess.

  • Estrone (E1) Test

Estrone E1 is the main type of estrogen found in men. It is produced by the adrenals and the testis. Fat cells also produce some amounts of estrogen. It is a weaker form of estrogen as compared to the other two. When needed, this is converted to estrogen.

E1 test is ordered in case of delayed puberty in men. A higher level can be due to tumors of the testicles or adrenal glands. The normal level of estrone in adult males is 10-60 pg/mL.

  • Estradiol (E2) Test

Estradiol is the predominant form of estrogen. In men, the adrenal glands and the testis make estradiol. Estradiol is the form of estrogen that regulates libido, erection, and sperm production in men (12).

Estradiol also regulates the production of testosterone. It inhibits as well as stimulates T-production. High estrogen can inhibit the functions of the hypothalamus and the pituitary. This can lead to a decrease in LH and FSH hormones ultimately resulting in low T (13). The normal level of estradiol in adult males is 10-40 pg/mL.

Estradiol E2 blood tests help the doctor to diagnose the cause of low T much more specifically and detect whether the problem is because of an estrogen excess of testosterone or an androgen deficiency. Low E2 levels lead to late puberty while higher levels lead to early puberty.

These tests are also used for checking estrogen levels during testosterone therapy.


DHEA stands for dehydroepiandrosterone. It is mainly produced by the adrenals. DHEA can be converted to testosterone as well as estradiol in men and women. In case, there are low levels of DHEA, the test helps to check whether there is a problem with your adrenals. 


DHEA is converted to DHEA-S in the adrenals and the liver. A high DHEA-S level can be due to cancer or tumors in the adrenals or can suggest overactive adrenals. A lower level means damaged adrenals or hypopituitarism. The test therefore can help detect the condition of the adrenal and the cause of low T.

Androstenedione Test

In case, there are abnormalities in other tests such as DHEA-S, testosterone, FSH, LH, prolactin, and estrogen, then androstenedione testing can help determine the cause of the testosterone imbalance. Androstenedione is a hormone produced by the adrenal glands and gonads. It is then converted to estrogen or testosterone as needed (14).

So androstenedione testing is commonly used to evaluate the condition of the gonads and the adrenals. It also helps determine the cause of high testosterone in women. This is mainly the case when a woman shows symptoms such as hirsutism, irregular periods or no periods, and infertility.

In boys with very early puberty, this test is recommended. This test is recommended only when there is an abnormality in testosterone levels.

Blood Tests of Testosterone Metabolites

When low T is diagnosed, measuring testosterone metabolites help evaluate the overall androgen activity. Too much or too little production of these steroid hormones can cause many health issues.

Even if testosterone levels are low, sufficient androgen activity is reassuring. Testosterone replacement treatments to individuals with low T but normal levels of total androgens may not be beneficial. 

What are Testosterone Metabolites: Male sex hormones are known as androgens. They play important roles in the development and regulation of sexual characteristics, in both men and women. The main androgens are testosterone and androstenedione. 

Testosterone is synthesized from cholesterol. Testosterone metabolism produces by-products (metabolites) such as Dihydrotestosterone (DHT) and Androstanediol Glucuronide. These testosterone metabolites also display androgen activity.

Androstanediol Glucuronide Test

Testing androstanediol glucuronide levels in the blood help evaluate how much androgen activity is taking place (15). This test accurately reflects the metabolic effects of dihydrotestosterone in target cells

Dihydrotestosterone (DHT) Test

DHT is one of the most potent androgens in the body. It is formed with the help of the enzyme 5-alpha-reductase (16). DHT is formed in the prostate glands, hair follicles, and adrenal glands.

A therapeutic level of testosterone is not enough to ensure androgen function. Appropriate DHT levels and activities on the other hand provide the essential masculinization that makes men look and behave like men.

The DHT test serves as the marker of testosterone production from peripheral tissue such as skin and the liver. It can also be a diagnostic tool in understanding if there are any prostate problems.

Salivary Test for Low T

As the name suggests, salivary-free testosterone measures the amount of free testosterone available in the body. This test does not get affected by the variations in the proteins that testosterone binds to.

Salivary tests for low T are a faster and easier way to check bioactive T-levels. However, they may be less accurate in comparison to serum tests. Salivary tests are non-invasive procedures. 

Urine Tests for Low T in Men

Testosterone levels in the blood are prone to variation in the day. Normally it is the highest in the morning. On the other hand, urinary testosterone levels are much more constant. They can be used for detecting abnormalities in testosterone production.

The following are the urine tests for checking testosterone levels:

  • Free testosterone: Urinary measurement of free testosterone, in a 24-hour urine sample, is a better indicator of testosterone deficiency if any. 
  • 17-Ketosteroids urine test: 17-ketosteroids are formed from the breakdown of sex hormones. This test is usually done on a 24-hour urine sample. This means you need to collect your urine samples for over one whole day. As these sex hormones are produced by the gonads and the adrenals, an increased level detects a tumor in the adrenal, testis, and/or ovary. They can be either benign or malignant. An overactive thyroid can also result in high 17-ketosteroids. Low levels indicate less than normal functioning of the adrenals and pituitary. 
  • Androsterone: Androsterone is a metabolite of testosterone and dihydrotestosterone (DHT). It accurately reflects the metabolic effects of dihydrotestosterone in the target cells. Urinary androsterone test is done, in a 24 hr urine sample, as a means to detect the cause of any development problems in puberty. 
  • Etiocholanolone: This is a metabolite of DHEA. It is excreted in the urine and Etiocholanolone testing is mostly done in females. These tests help to evaluate the cause of high testosterone in women. Along with the DHEA tests, Etiocholanolone is tested in women with symptoms such as thinning hair, acne, and mood disorders.

Imaging tests

Imaging tests for diagnosing low testosterone include imaging of the prostate, doppler of carotid arteries, and checking on the bone density using DEXA.

  • MRI – MRI (Magnetic Resonance Test) is done to check on the condition of the pituitary and the hypothalamus (17). This can help identify whether it is primary or secondary hypogonadism.
  • Bone age estimation to check on the bone density (DEXA): Low testosterone often leads to decreased bone density and reduced bone mass. A dual-energy x-ray absorptiometry (DEXA) accurately confirms the density of the bone (18).
  • Doppler of the carotid artery: The carotid arteries are situated on both sides of the neck. These arteries are the main blood vessels that deliver blood to your brain. Long-term testosterone deficiency increases your risk of clogged arteries including carotid arteries (19). Doppler of the carotid artery is an imaging test that can detect this. 
  • Prostate Imaging for checking for prostate cancer: Long-term testosterone deficiency has a direct impact on the prostate glands. Prostate imaging helps detect BPH (benign prostate enlargement) and cancer.
  • Dynamic uroflowmetry: This is done to assess the urinary flow. Both the speed and volume of urine are measured. An abnormal flow suggests a block in the urine flow or the weak muscles of your bladder. 

The Final Note

Accurate detection of abnormal testosterone levels can help prevent many problems, including delayed puberty.

Early diagnosis can help your doctor find the cause of low testosterone and start treatment accordingly. It will offer better protection against long-term complications of low T such as infertility, osteoporosis, metabolic syndrome, and cardiovascular problems.

Read more: The Consequences of Long Term Untreated Low T

About Anju Mobin 104 Articles
The Nutritionist Wordsmith with a Chocoholic Edge! Anju is not just a writer; she's a brush-wielding, coffee-sipping, chocolate-loving wordsmith! With a Fine Arts degree in one hand and a Home Science degree in the other, she blends creativity and nutrition to craft content that truly nourishes the mind. Whether she's painting a vivid picture with words or brewing up engaging web content, she pours her heart into every project. When she's not busy whipping up articles, you'll find her nurturing orchids to bloom in her garden, baking scrumptious treats, and indulging her chocoholic tendencies with dark delights. She is the founder and managing editor of Fitness Hacks.