What is the difference between free testosterone and total testosterone?

Understanding Free Testosterone versus Total Testosterone

Total testosterone measures all testosterone in the blood (both bound and unbound), while free testosterone measures only the biologically active, unbound fraction that can enter cells and exert hormonal effects—free testosterone is the metabolically active form and provides a more accurate assessment of true androgen status, especially when binding protein levels are altered. 1

Key Physiological Differences

Total Testosterone

• Represents the sum of all testosterone in circulation, including hormone bound to proteins and the small unbound fraction 2
• Normal range in adult men is 300-800 ng/dL in most laboratories 1
• Approximately 98% is protein-bound: tightly bound to sex hormone-binding globulin (SHBG) or weakly bound to albumin 1
• Can be misleading when SHBG levels are altered by obesity, age, medications, hyperthyroidism, or other conditions 1, 3

Free Testosterone

• Represents only the 2% unbound fraction that is biologically active and can diffuse into cells 4, 2
• This is the metabolically active fraction that exerts androgenic effects on target tissues 2, 5
• Provides superior diagnostic accuracy compared to total testosterone, particularly in conditions affecting binding proteins 1, 6
• Essential for accurate assessment in men with obesity who may have low total testosterone solely due to low SHBG but normal free testosterone 1

Clinical Significance: When Free Testosterone Matters Most

In Men with Obesity

Men with obesity frequently have low total testosterone due to decreased SHBG concentrations, but their free testosterone may be normal 1. This occurs because:

• Obesity-related decreases in testosterone are often attributable to low SHBG 1
• When low total testosterone is due solely to low SHBG, free testosterone levels remain normal 1
• A subset will have frankly low free testosterone due to increased aromatization of testosterone to estradiol in adipose tissue 1

Therefore, measuring morning serum free testosterone is essential when evaluating low total testosterone in men with obesity 1

In Women with Androgen Excess

Free testosterone measurement is superior for diagnosing hirsutism and polycystic ovary syndrome (PCOS) 2, 6. Evidence shows:

• Each of 32 hirsute women studied had significantly elevated free testosterone, although half had total testosterone within normal limits 6
• Elevation of free testosterone is the hallmark of hirsutism, making it clearly superior to total testosterone for identification 6
• Recent PCOS guidelines emphasize the diagnostic challenges of biochemical hyperandrogenism assessment 1

In Conditions Affecting Binding Proteins

Free testosterone provides more accurate assessment when SHBG or albumin levels are altered 1:

• Hyperthyroidism (increases SHBG)
• Liver disease (decreases albumin)
• Medications (oral estrogens increase SHBG)
• Aging (SHBG increases with age)

Measurement Methods: Critical Considerations

Gold Standard for Free Testosterone

Equilibrium dialysis is the reference standard method for measuring free testosterone 1. This method:

• Directly measures the unbound fraction
• Is technically complex and not widely available in routine laboratories 1
• Should be used when accurate free testosterone measurement is needed 1

Alternative Approaches

Calculated Free Testosterone:

• Can be estimated using total testosterone, SHBG, and albumin levels 1, 3
• Free androgen index (FAI) = total testosterone ÷ SHBG × 100 1
• Calculations are prone to error due to assumptions about binding constants 3
• FAI is not recommended for use in men due to inaccuracy at extremes of SHBG concentration 3

Direct Immunoassays:

• Available at most local laboratories but have limited reliability 1
• Analog methods for free testosterone are particularly problematic 1
• Should be avoided when accurate assessment is critical 1

Recommended Clinical Approach

For Suspected Hypogonadism in Men

Obtain morning (8-10 AM) total testosterone first, then add free testosterone by equilibrium dialysis if total testosterone is low 1:

1. Morning total testosterone measurement (8-10 AM)
2. If low, confirm with repeat testing due to assay variability 1
3. Measure free testosterone by equilibrium dialysis AND SHBG in patients with obesity or conditions affecting binding proteins 1
4. If both are low on at least 2 separate assessments, proceed with hypogonadism workup 1

For Suspected Androgen Excess in Women

Free testosterone measurement is essential and superior to total testosterone alone 6:

• Provides better identification of hirsutism and PCOS
• Detects androgen excess even when total testosterone is normal
• Should be measured using reliable assays (equilibrium dialysis preferred) 1

Common Pitfalls to Avoid

• Do not rely on total testosterone alone in patients with obesity, liver disease, thyroid disorders, or those taking medications affecting SHBG 1, 3
• Avoid direct immunoassay methods for free testosterone when accurate assessment is needed—they have limited reliability 1
• Do not use FAI in men—it is inaccurate at extremes of SHBG concentration 3
• Always draw morning samples (8-10 AM) for testosterone testing due to diurnal variation 1
• Confirm abnormal results with repeat testing before initiating treatment 1