Testosterone: Blood Test, Norms, and Causes of Deviations

Testosterone is the primary androgen in men, but it is also present in women and children. In men, most of the hormone is produced in the Leydig cells of the testes under the influence of luteinizing hormone, while in women, testosterone is synthesized in the ovaries and adrenal glands and is formed from the peripheral conversion of other steroids. Therefore, testosterone testing is not a "male-only" test. It is also prescribed for suspected hypogonadism in men, hyperandrogenism in women, and pediatric endocrine conditions. [1]

Testosterone exists in more than one form in the blood. Most of it is bound to sex hormone-binding globulin and albumin. The free fraction is very small, typically less than 2% of the total, but it is considered the most biologically active. Because of this, normal total testosterone does not always indicate normal androgen status, and low total testosterone does not always indicate true deficiency. [2]

Total testosterone is used in most cases for the initial evaluation of men. This is the recommended initial test in both the Endocrine Society and the European Association of Urology guidelines. Free testosterone is assessed when total testosterone is in the borderline range or when conditions alter sex hormone-binding globulin levels. [3]

It's important to distinguish between two analytical goals. The first is to detect testosterone deficiency in symptomatic men. The second is to detect androgen excess in women, primarily with hirsutism, acne, androgenetic alopecia, menstrual irregularities, or virilization. These two situations require different diagnostic approaches, different thresholds, and often different methodological requirements. [4]

Therefore, a testosterone test cannot be viewed as a simple number on a form. Proper interpretation requires answers to four questions: who is being tested, why, what method is used to measure the hormone, and whether the results are affected by sex hormone-binding globulin, age, medications, or hormonal contraception. Without these answers, even a technically correct test can easily lead to an erroneous conclusion. [5]

Table 1. Which forms of testosterone are important in clinical practice

Indicator	What does it mean?	When it is especially useful
Total testosterone	The sum of bound and free testosterone	Initial testing in men with suspected hypogonadism
Free testosterone	Biologically active unbound fraction	Borderline total testosterone, altered sex hormone-binding globulin levels
Sex hormone-binding globulin	A protein that binds a significant portion of testosterone	Helps to understand why total testosterone does not match clinically
Albumen	A protein with weaker testosterone binding	Used in the calculation of free testosterone
Estimated free testosterone	Calculated from total testosterone, sex hormone-binding globulin, and albumin	Preferred over direct, inaccurate free testosterone immunoassays

Source for the table: [6]

When is a testosterone test really necessary?

In men, testing is primarily warranted if they have symptoms that genuinely increase the likelihood of androgen deficiency. The most specific symptoms include decreased libido, decreased morning erections, erectile dysfunction, infertility, bone loss, unexplained anemia, severe fatigue, and decreased muscle mass. Current guidelines emphasize that routine screening of asymptomatic men is unnecessary. [7]

In women, the test is not prescribed to detect "low testosterone for the sake of well-being," but primarily when hyperandrogenism is suspected. This includes hirsutism, adult acne, androgenic hair loss, irregular menstruation, amenorrhea, infertility, and rapidly increasing signs of virilization. In this group, the test is especially valuable when clinical manifestations are minimal or ambiguous. [8]

In children and adolescents, testing is prescribed only for clear indications: precocious or delayed puberty, suspected congenital or acquired gonadal dysfunction, intersex conditions, tumors, or pituitary disorders. In these cases, the results must be correlated with age and stage of puberty. Adult norms should not be used as a guide in pediatrics. [9]

There's a downside, though. The test isn't necessary as a "just in case" screening for common fatigue without specific symptoms, a one-time mood swing, or simply out of interest in staying in shape. Low testosterone levels without characteristic clinical symptoms and without repeated confirmation are not a diagnosis of hypogonadism. The Endocrine Society particularly emphasizes this. [10]

Fertility is also a key consideration. If a man complains of infertility, testosterone alone is not enough. At least luteinizing hormone, follicle-stimulating hormone, and often a sperm analysis are needed. In cases of proven hypogonadism, treatment is especially important, as exogenous testosterone can suppress spermatogenesis. [11]

Table 2. When the test is especially informative

Group	The most typical indications
Men	Decreased libido, erectile dysfunction, infertility, osteopenia, anemia, suspected hypogonadism
Women	Hirsutism, acne, alopecia, menstrual irregularities, infertility, virilization
Children and teenagers	Precocious puberty, delayed puberty, suspected gonadal or pituitary pathology
Patients with altered sex hormone-binding globulin	Discrepancy between symptoms and total testosterone
Patients on hormonal therapy or with suspected tumor	Monitoring and clarification of the source of androgens according to indications

Source for the table: [12]

How to Properly Take a Testosterone Blood Test

Men are recommended to have their total testosterone levels tested in the morning, when hormone levels are typically highest. MedlinePlus recommends testing between 7 and 10 a.m., while the European Association of Urology recommends testing between 7 and 11 a.m., preferably on an empty stomach. If the first result is low, it should be confirmed with a repeat test on another day. [13]

The requirement for repeat testing is one of the most important principles of modern diagnostics. The Endocrine Society, the American Urological Association, and the European Association of Urology all recognize that hypogonadism cannot be diagnosed based on a single measurement. Men require two low morning results, combined with symptoms. [14]

The method of measurement is important. For women, children, and men with very low levels, liquid chromatography with tandem mass spectrometry is preferred, as conventional immunoassays are less accurate at low concentrations. For free testosterone, equilibrium dialysis or corrected calculation are preferred, while direct immunoassays for free testosterone are considered unreliable. [15]

It is important for women to consider hormonal contraception. Combined oral contraceptives increase sex hormone-binding globulin levels and reduce gonadotropin-dependent androgen production, making it difficult to reliably assess biochemical hyperandrogenism. If androgen assessment is essential, international guidelines for polycystic ovary syndrome recommend discontinuing the use of combined oral contraceptives for at least 3 months with alternative contraception. [16]

Another practical consideration concerns biotin. Normal dietary amounts of biotin, obtained from food and standard multivitamins, generally do not distort results. However, high doses of supplements, starting from approximately 5 mg and above, can interfere with some biotinylated immunoassays. Therefore, it is important to inform your doctor and the laboratory about your biotin intake before donating blood. [17]

For follow-up, it is advisable to have the test performed in the same laboratory and, if possible, using the same method. This is especially important for borderline values, in women, and in adolescents, when even moderate analytical differences between platforms can alter the interpretation. [18]

Table 3. Preparation for testosterone testing

Question	Practical recommendation
When to hand over to men	In the morning, from about 7 to 11 o'clock
Is a repeat analysis necessary?	Yes, if the first result is low
Is a fasting interval necessary?	Preferably on an empty stomach, especially for men
Which method is better for women and children?	Liquid chromatography with tandem mass spectrometry
How to evaluate free testosterone	By calculation or by equilibrium dialysis method
What to do with biotin	Report additives, especially at high doses
Is it possible to evaluate androgens in the context of combined oral contraceptives?	This is not reliable for accurately assessing hyperandrogenism.

Source for the table: [19]

Testosterone levels and how to read the results correctly

The concept of "normal testosterone" is much more complex than it seems. In men, the laboratory reference interval and the diagnostic threshold for hypogonadism do not completely coincide. The Harmonized Reference Range, obtained using standardized methods in a population of healthy men aged 19-39 with a body mass index less than 30, is 264-916 ng/dL. This range is useful as a guide, but a diagnosis of hypogonadism still requires symptoms and repeated confirmation. [20]

The American Urological Association suggests using a total testosterone level below 300 ng/dL as a reasonable diagnostic threshold to support a diagnosis, and the European Association of Urology considers 12 nmol/L as a reliable working threshold for late-onset hypogonadism in symptomatic men. These values appear different, but in real-world practice, they describe a close "borderline" zone and should always be interpreted in conjunction with symptoms and repeat testing. [21]

Free testosterone levels aren't necessary for everyone. Their assessment is particularly useful when total testosterone is at the lower limit of normal and clinical findings are compelling, or when sex hormone-binding globulin levels fluctuate. The European Association of Urology indicates that a calculated free testosterone level below 220 pmol/L may increase the likelihood of a correct diagnosis of hypogonadism, especially with a total testosterone level in the range of 8-11 nmol/L. However, this is not a universal, standalone threshold, but rather a supporting guideline. [22]

The situation is even more complex for women and children. The international guidelines for polycystic ovary syndrome explicitly emphasize that reference intervals vary significantly between laboratories and methods, and direct immunoassays at low concentrations are not sufficiently accurate. Therefore, male thresholds cannot be used for women, and results from different laboratories cannot be compared without adjusting for the method. [23]

In pediatrics, normal values depend not only on age but also on the stage of puberty. For example, in boys at stage 5 of puberty, the range may be comparable to adult values, while in girls at the same stage, it may be tens of times lower. This is another reason why a child's results should be assessed by a pediatric endocrinologist, rather than using a generic reference from the internet. [24]

Finally, testosterone levels should be closely related to clinical findings. A man with a total testosterone level of 290 ng/dL and typical symptoms requires a completely different approach than a man with the same level but no complaints. Similarly, in women, a moderate increase in testosterone, accompanied by slowly developing hirsutism and irregular cycles, is more often consistent with polycystic ovary syndrome, whereas rapidly increasing virilization requires a search for a tumor. [25]

Table 4. Examples of reference intervals and diagnostic benchmarks

Indicator	Landmark
Standardized range of total testosterone in healthy men aged 19-39 years	264-916 ng per dl
American Urological Association diagnostic threshold for male hypogonadism	below 300 ng/dl
European Association of Urology working threshold for symptomatic late hypogonadism	below 12 nmol/L
A possible auxiliary reference value for estimated free testosterone in men	below 220 pmol per l
Example of laboratory range in adult premenopausal women using liquid chromatography-tandem mass spectrometry	10-55 ng per dl
Example of laboratory range in adult postmenopausal women by liquid chromatography-tandem mass spectrometry	7-40 ng per dl

Source for the table: [26]

Table 5. Examples of pediatric ranges by pubertal stage

Group	Example of a total testosterone range
Boys, stage 1	less than 2.5-10 ng per dl
Boys, stage 2	18-150 ng per dl
Boys, stage 3	100-320 ng per dl
Boys, stage 4	200-620 ng per dl
Boys, stage 5	350-970 ng per dl
Girls, stage 5	20-38 ng per dl

Source for the table: [27]

Why testosterone levels are low

The most common cause of moderately low testosterone in adult men is not a rare tumor, but rather functional suppression of the hypothalamic-pituitary-gonadal axis due to obesity, type 2 diabetes, chronic diseases, obstructive sleep apnea, certain medications, and general metabolic dysfunction. The European Association of Urology emphasizes the high prevalence of low testosterone in these groups. [28]

There are two broad categories of hypogonadism. In primary hypogonadism, the problem is in the testicles, so testosterone is low, and luteinizing hormone and follicle-stimulating hormone are usually elevated. In secondary hypogonadism, the hypothalamus or pituitary gland is affected, so testosterone is low, and gonadotropins are low or abnormally normal. This is why, after low testosterone is confirmed, luteinizing hormone and follicle-stimulating hormone are always needed. [29]

Classic causes of primary hypogonadism include Klinefelter syndrome, previous orchitis, trauma, toxic injury, the effects of chemotherapy, radiation therapy, and age-related irreversible changes in testicular tissue. Secondary hypogonadism may be associated with hyperprolactinemia, pituitary tumors, systemic diseases, obesity, opioid and glucocorticoid use. [30]

Sex hormone-binding globulin plays a special role. If it is reduced, for example in obesity, hypothyroidism, or insulin resistance, total testosterone may be low, although free testosterone remains relatively intact. Conversely, with high levels of this protein, total testosterone may appear more healthy than the hormone actually available to tissues. This is why free testosterone assessment is necessary in complex cases. [31]

Very low values require a more cautious search for an organic cause. The Endocrine Society and the European Association of Urology recommend additional testing to clarify the cause, and in secondary hypogonadism with severely decreased testosterone and signs of pituitary pathology, magnetic resonance imaging of the pituitary region is considered. [32]

Why testosterone levels are elevated

In men, moderately elevated testosterone levels are more often associated not with disease, but with external administration of androgens, anabolic steroids, or replacement therapy. Much less commonly, testicular tumors, adrenal tumors, or rare forms of congenital pathology are the cause. Therefore, a high result without information about the medications used may be misinterpreted. [33]

In women, elevated testosterone is significantly more clinically important because even moderate androgen excess can be accompanied by hirsutism, acne, menstrual irregularities, and infertility. The most common cause is polycystic ovary syndrome. However, with the rapid onset of virilization, deepening of the voice, clitoromegaly, a sharp increase in muscle mass, or very rapid progression of symptoms, it is necessary to look for an androgen-producing tumor and ovarian hyperthecosis. [34]

The assessment of elevated testosterone in women should always take into account the method. The International Guidelines for Polycystic Ovary Syndrome recommend starting with total and free testosterone, measured using high-precision methods. If these are not elevated but clinical suspicion remains, androstenedione and dehydroepiandrosterone sulfate may be considered, but their specificity is lower. [35]

In childhood, elevated testosterone levels require particularly careful assessment. They may reflect early puberty, congenital adrenal hyperplasia, tumors, exposure to exogenous androgens, or laboratory confusion with age-appropriate reference values. Because normal values change rapidly during puberty, a high result alone does not establish a diagnosis. [36]

False alarms should also be considered. Sometimes a result appears "high" simply because it is compared to an inappropriate interval, for example, a female result is compared to a male one, an adolescent result to an adult one, or a liquid chromatography-tandem mass spectrometry result is compared to an old immunoassay reference. Such errors are more common than rare androgen-producing tumors. [37]

Table 6. Main reasons for decrease and increase of testosterone

Change	Common causes
Decrease in men	Obesity, type 2 diabetes, chronic diseases, opioids, glucocorticoids, Klinefelter syndrome, testicular disease, hyperprolactinemia, pituitary pathology
Increase in men	Exogenous testosterone, anabolic steroids, and, less commonly, testicular and adrenal tumors
Increase in women	Polycystic ovary syndrome, ovarian hyperthecosis, ovarian or adrenal tumors, and, less commonly, drug-induced causes
Decrease in women	In isolation it is of little diagnostic value and is interpreted only in a clinical context.
Increase in children	Precocious puberty, congenital adrenal hyperplasia, exogenous androgens, tumors

Source for the table: [38]

What additional tests are needed after testosterone abnormalities?

If low testosterone is confirmed in a man, the next step is usually testing luteinizing hormone and follicle-stimulating hormone. These tests help determine whether the condition is primarily testicular or secondary hypogonadism. In the secondary case, prolactin and other pituitary hormones are often added. [39]

If there is a suspicion of sex hormone-binding globulin (SHBG) influence, this protein should be measured and free testosterone recalculated. This is especially useful in cases of obesity, liver disease, thyroid disease, estrogen use, anticonvulsants, and old age. Without this, borderline total testosterone levels are often misinterpreted. [40]

In men with infertility, not only hormones but also a spermogram are important. Men with significantly low testosterone, secondary hypogonadism, elevated prolactin, or signs of a space-occupying lesion may require magnetic resonance imaging of the pituitary gland. This approach is clearly reflected in the recommendations of the Endocrine Society and the European Association of Urology. [41]

In women, after hyperandrogenism is confirmed, dehydroepiandrosterone sulfate, androstenedione, 17-hydroxyprogesterone, prolactin, and thyroid-stimulating hormone are additionally assessed, and, if necessary, ovarian and adrenal imaging is performed. The specific test depends on the speed of symptom onset and the clinical profile. When symptoms progress slowly and ovulation disorders are combined, polycystic ovary syndrome is more often suspected. [42]

In pediatrics, additional testing is almost always more extensive than in adults: bone age, gonadotropins, adrenal steroids, genetic testing as indicated, ultrasound, and assessment of the stage of puberty. This once again demonstrates that testosterone is only one piece of a larger endocrine picture, not a standalone diagnosis. [43]

FAQ

Should men always have their testosterone levels tested in the morning?

Yes, this is appropriate for an initial assessment in men. Testosterone levels have a circadian rhythm, and the general recommendations recommend taking blood samples in the morning, usually between 7 and 11 a.m. This reduces the risk of falsely low or difficult-to-interpret results. [44]

Is it possible to make a diagnosis based on one test?

No. Current guidelines require symptoms and two confirmed low morning results. One test may be transiently low or just borderline. [45]

What is more important - total testosterone or free testosterone?

For most men, total testosterone is the starting test. Free testosterone is especially important for borderline values and conditions that alter sex hormone-binding globulin levels. In women and children, with low concentrations, not only the choice of testosterone but also the accurate measurement method is crucial. [46]

Is it possible to take the test if you are obese?

It's possible, but interpretation must take into account that obesity often reduces sex hormone-binding globulin levels, which can cause total testosterone to appear lower than actual androgen status. In such cases, measuring sex hormone-binding globulin and calculating free testosterone are useful. [47]

Can women take testosterone tests while taking combined oral contraceptives?

This is a poor option for accurately assessing biochemical hyperandrogenism. These drugs increase levels of sex hormone-binding globulin and suppress androgen production, so international guidelines recommend a 3-month suspension with alternative contraception if testing is truly necessary. [48]

Why does the lab say "normal" but there are symptoms?

Because a laboratory reference value and a clinical diagnosis are not the same thing. The result may be borderline, total testosterone may not reflect free testosterone, and symptoms may be caused not only by androgen deficiency but also by other conditions: depression, sleep apnea, hyperprolactinemia, chronic diseases, iron deficiency, and diabetes. [49]

Is testosterone necessary for infertility?

Yes, but not as the only test. For male infertility, it is assessed along with luteinizing hormone, follicle-stimulating hormone, and a spermogram. In women, testosterone levels may be increased as part of an evaluation for anovulation and polycystic ovary syndrome. [50]

Can biotin spoil the results?

Yes, at high doses, this is possible for some immunoassays. Normal dietary amounts are usually not a problem, but supplementation with 5 mg or more can cause analytical distortions. Therefore, biotin intake must be reported before testing. [51]

Conclusion

A blood test for testosterone is not a simple "male potency" test or a universal fatigue test. It is a contextual hormonal analysis, the meaning of which depends on gender, age, symptoms, time of collection, measurement method, and sex hormone-binding globulin levels. It is especially important to distinguish between the male diagnosis of testosterone deficiency and the female diagnosis of hyperandrogenism, as the algorithms for both are different. [52]

The main rule of thumb is that hypogonadism in men is not confirmed based on a single result, and in women, testosterone testing cannot be reliably interpreted without an accurate method and consideration of hormonal contraception. The more accurate the pre-analysis and the more carefully the additional tests are selected, the more useful the result will be and the lower the risk of misdiagnosis. [53]