What is the difference between testosterone and free testosterone?

Understanding the Difference Between Testosterone and Free Testosterone

Total testosterone measures all testosterone in your blood (both bound and unbound), while free testosterone measures only the biologically active fraction that is not bound to proteins—and free testosterone is what actually matters for diagnosing hormone disorders. 1, 2

What Each Measurement Represents

Total Testosterone

• Total testosterone includes all testosterone circulating in your bloodstream, regardless of whether it can actually be used by your body 2
• Approximately 98% of total testosterone is bound to proteins: 60-70% tightly bound to sex hormone-binding globulin (SHBG) and 30-40% weakly bound to albumin 1, 2
• Only about 2% circulates as free (unbound) testosterone 1, 3

Free Testosterone

• Free testosterone is the unbound fraction that can enter cells and exert biological effects 1, 2
• This is the metabolically active form responsible for maintaining libido, muscle mass, bone density, and other androgen-dependent functions 2
• According to the free hormone hypothesis, biological activity of testosterone is best reflected by free rather than total hormone concentrations 1

Why This Distinction Matters Clinically

Diagnostic Accuracy

• In men with borderline-low total testosterone (around 300-350 ng/dL), measuring free testosterone prevents both under-diagnosis and over-diagnosis of hypogonadism 1, 4
• Changes in SHBG levels (caused by obesity, aging, diabetes, or medications) can significantly alter total testosterone while free testosterone remains normal—or vice versa 1, 5
• The 2023 International PCOS Guidelines recommend using both total testosterone and free testosterone as first-line tests for diagnosing biochemical hyperandrogenism, with free testosterone showing superior sensitivity (89%) compared to total testosterone (74%) 6

Common Clinical Scenarios Where Free Testosterone Is Essential

Obesity and metabolic syndrome: Low SHBG artificially lowers total testosterone, but free testosterone may be normal—measuring only total testosterone would lead to overdiagnosis of hypogonadism 7, 4

Elderly men: SHBG increases with age, potentially elevating total testosterone while free testosterone remains low—measuring only total testosterone would miss true hypogonadism 7, 1

Borderline total testosterone (275-350 ng/dL): Free testosterone measurement determines whether true biochemical hypogonadism exists 7, 4

How Free Testosterone Should Be Measured

Gold Standard Method

• Equilibrium dialysis coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the reference standard for measuring free testosterone 6, 2
• This method physically separates free testosterone from protein-bound testosterone before measurement 2
• Unfortunately, equilibrium dialysis is too complex and expensive for routine clinical use 5, 2

Practical Clinical Alternatives

Calculated free testosterone using the Vermeulen equation is the preferred method when equilibrium dialysis is unavailable, requiring measurement of total testosterone, SHBG, and albumin 6, 3, 5

Free androgen index (FAI) can be used as an alternative, calculated as (total testosterone/SHBG) × 100, though it has limitations at extremes of SHBG concentration 6, 5

Direct immunoassays for free testosterone are inaccurate and should not be used—they show spurious correlations with SHBG and misclassify androgen status 5, 2, 8

Critical Pitfalls to Avoid

• Never rely on total testosterone alone when SHBG is abnormal (high in elderly men, low in obese men with metabolic syndrome) 7, 1
• Never use direct immunoassay methods for free testosterone—they are fundamentally flawed and produce misleading results 5, 2, 8
• Never assume that normal total testosterone excludes hypogonadism in men with low SHBG—26.3% of men over 60 have normal total testosterone but low free testosterone 7
• Never diagnose hypogonadism based on a single testosterone measurement—always confirm with repeat morning measurements on separate occasions 7, 4

Recommended Diagnostic Approach

For suspected male hypogonadism: Measure morning total testosterone (8-10 AM) on two separate occasions; if borderline or low, add free testosterone by equilibrium dialysis or calculated method, plus SHBG 7, 4

For suspected PCOS/hyperandrogenism in women: Use both total testosterone and free testosterone as first-line tests, preferably measured by LC-MS/MS 6

When interpreting results: Free testosterone provides the most accurate assessment of biologically active androgen status, particularly when SHBG is abnormal or total testosterone is borderline 1, 2