How do total testosterone and free testosterone differ, and in which clinical situations should free testosterone be measured instead of total testosterone?

Total Testosterone vs Free Testosterone: Clinical Differences and When to Measure Each

Measure free testosterone by equilibrium dialysis (or calculate it using validated formulas) whenever total testosterone is borderline (230–350 ng/dL), in men with obesity, or in any condition that alters sex hormone-binding globulin (SHBG)—because total testosterone alone will mislead you in these situations. 1

Key Physiological Differences

Total testosterone represents all circulating testosterone: approximately 98% is protein-bound (60% tightly to SHBG, 38% weakly to albumin), leaving only 2% as free testosterone 1, 2. The normal range for total testosterone in adult men is 300–800 ng/dL in most laboratories 1.

Free testosterone is the unbound, biologically active fraction that enters cells and exerts androgenic effects 1, 2. It provides superior diagnostic accuracy when SHBG levels are altered by obesity, aging, medications, hyperthyroidism, liver disease, or other conditions 1.

Why Total Testosterone Can Be Misleading

• Obesity is the most common clinical scenario where total testosterone misleads: men with obesity frequently have low total testosterone solely due to decreased SHBG concentrations, but their free testosterone may be completely normal 1
• Obesity-related decreases in testosterone are often attributable to low SHBG rather than true androgen deficiency 1
• Conversely, conditions that elevate SHBG (hyperthyroidism, aging, liver disease, certain medications) can produce normal or high-normal total testosterone while free testosterone remains frankly low 1

Clinical Situations Requiring Free Testosterone Measurement

1. Borderline Total Testosterone (230–350 ng/dL)

• When total testosterone falls in the "gray zone," measuring free testosterone by equilibrium dialysis is essential to distinguish true hypogonadism from SHBG-related variation 1, 3
• Total testosterone between 280–350 ng/dL is not sensitive enough to reliably exclude hypogonadism; it must exceed 350–400 ng/dL to reliably predict normal free testosterone 3
• At total testosterone <350 ng/dL, sensitivity for detecting low free testosterone is only 96.8%, meaning you will miss cases without direct free testosterone assessment 3

2. Obesity

• This is the single most important indication for free testosterone measurement 1
• Men with obesity may have low total testosterone due to decreased SHBG but normal free testosterone—meaning they do not have true hypogonadism and should not receive testosterone therapy 1
• Measuring morning serum free testosterone is essential when evaluating low total testosterone in men with obesity 1

3. Conditions Affecting SHBG

Free testosterone measurement is superior and necessary in:

• Aging (SHBG increases with age) 1
• Liver disease (cirrhosis elevates SHBG) 1
• Hyperthyroidism (increases SHBG) 1
• Medications that alter SHBG (anticonvulsants, estrogens, thyroid hormone) 1
• Diabetes (often associated with low SHBG) 1

4. Women with Suspected Androgen Excess

• Free testosterone measurement is essential and superior to total testosterone alone for diagnosing hirsutism and polycystic ovary syndrome (PCOS) 1

Measurement Methods: Critical Technical Considerations

Gold Standard: Equilibrium Dialysis

• Equilibrium dialysis is the reference standard method for measuring free testosterone 1, 2
• This method physically separates free from bound testosterone and provides the most accurate measurement 2
• It is technically demanding and expensive, limiting routine clinical availability 2, 4

Calculated Free Testosterone

• Can be estimated using total testosterone, SHBG, and albumin levels with validated formulas (e.g., Vermeulen equation) 1, 2, 5
• Calculated free testosterone is prone to error due to assumptions about binding constants and variations in albumin 1, 6
• A fixed albumin of 4.3 g/dL is acceptable for most clinical evaluations, but accuracy decreases when albumin is ≤3.5 g/dL combined with SHBG ≤30 nmol/L (occurs in 1.2% of cases) 6
• When albumin is low and SHBG is low, equilibrium dialysis measurement is warranted for better accuracy 6

Direct Immunoassays: Avoid When Accuracy Matters

• Direct immunoassays for free testosterone have limited reliability and should be avoided when accurate assessment is critical 1, 2
• These analog methods do not physically separate free from bound testosterone and are subject to significant interference 2

Recommended Clinical Approach: A Practical Algorithm

Step 1: Initial Assessment

• Obtain morning (8–10 AM) total testosterone first on two separate occasions 1, 7
• If both values are <300 ng/dL, proceed with hypogonadism workup 1

Step 2: Add Free Testosterone in Specific Situations

Measure free testosterone by equilibrium dialysis (or calculate it) when:

• Total testosterone is 230–350 ng/dL (borderline) 1, 3
• Patient has obesity 1
• Patient has conditions affecting SHBG (liver disease, thyroid disorders, medications) 1
• You are evaluating a woman for androgen excess 1

Step 3: Measure SHBG Concurrently

• Always measure SHBG alongside free testosterone to understand the binding protein context 1, 7
• The free androgen index (total testosterone ÷ SHBG) can provide additional diagnostic information 8, 5

Step 4: Confirm Abnormal Results

• If both total and free testosterone are low on at least 2 separate assessments, proceed with full hypogonadism workup (LH, FSH) 1

Common Pitfalls to Avoid

Do Not Rely on Total Testosterone Alone in High-Risk Populations

• Never use total testosterone alone in patients with obesity, liver disease, thyroid disorders, or those taking medications affecting SHBG 1
• Total testosterone <350 ng/dL lacks sufficient sensitivity to exclude hypogonadism without free testosterone assessment 3

Avoid Direct Immunoassay Methods

• Do not use direct immunoassay methods for free testosterone when accurate assessment is needed—they have limited reliability 1, 2
• Insist on equilibrium dialysis or validated calculated methods 1

Always Draw Morning Samples

• Always draw morning samples (8–10 AM) for testosterone testing due to diurnal variation 1, 7
• Afternoon or evening samples will be falsely low and lead to overdiagnosis 1

Confirm Before Treating

• Confirm abnormal results with repeat testing before initiating treatment 1
• Single measurements are insufficient due to assay variability and biological fluctuation 1

Do Not Ignore SHBG Context

• Measuring free testosterone without SHBG is incomplete—you need both to understand whether low total testosterone reflects true androgen deficiency or altered binding protein 1, 7