What is the difference between total testosterone and free testosterone in adult males with suspected hypogonadism?

Total Testosterone vs Free Testosterone in Suspected Hypogonadism

Total testosterone alone is insufficient to diagnose or exclude hypogonadism in adult males—free testosterone measurement is essential when total testosterone is borderline (280-400 ng/dL) or when conditions affecting sex hormone-binding globulin (SHBG) are present, particularly in men over 60 years, those with obesity, or diabetes. 1, 2, 3

Understanding the Key Differences

Total Testosterone

• Represents all testosterone in circulation: approximately 98% is bound to proteins (60% to SHBG, 38% to albumin), with only 2% circulating as free testosterone 1, 4
• Should be measured in the morning (8-10 AM) in a fasting state using liquid chromatography tandem mass spectrometry in a certified laboratory 1, 4
• Requires confirmation with a second measurement due to significant diurnal variation and assay variability 5, 4
• The threshold of <300 ng/dL is commonly used to define hypogonadism, though this misses a substantial proportion of men with true androgen deficiency 1, 2

Free Testosterone

• Represents the biologically active, unbound fraction that can enter cells and exert androgenic effects 1, 6
• Should be measured by equilibrium dialysis (gold standard) or calculated using validated equations (Vermeulen method preferred) with total testosterone, SHBG, and albumin 1, 6, 7
• More accurately reflects tissue-available testosterone, especially when SHBG is abnormal 1, 4
• A threshold of <6.5 ng/dL (or <63 pg/mL) indicates hypogonadism in most validated studies 3, 8

Critical Clinical Scenarios Where Free Testosterone is Mandatory

Men Over 60 Years

• 26.3% of men over 60 with erectile dysfunction have normal total testosterone but low free testosterone, representing symptomatic hypogonadism that would be completely missed by total testosterone screening alone 3
• SHBG increases with age, binding more testosterone and reducing the free fraction despite seemingly normal total levels 3, 8
• 74.2% of men with normal total testosterone but low free testosterone are over 60 years old, making age the primary risk factor for this discordance 3

Borderline Total Testosterone (280-400 ng/dL)

• Total testosterone between 280-350 ng/dL has only 91% sensitivity for detecting low free testosterone—meaning 9% of hypogonadal men are missed 2
• Total testosterone must exceed 350-400 ng/dL to reliably predict normal free testosterone with 96.8-98.2% sensitivity 2
• Only 24.7% of men with borderline total testosterone actually have confirmed hypogonadism when free testosterone is measured, preventing overtreatment 8

Obesity and Metabolic Conditions

• Men with obesity have increased aromatization of testosterone to estradiol in adipose tissue, causing secondary hypogonadism with disproportionately low free testosterone 1, 6
• Low SHBG in obesity artificially lowers total testosterone while free testosterone may be normal or only mildly reduced 1, 5
• Free testosterone measurement is essential in diabetic men with total testosterone near the lower limit of normal 1, 6

Conditions Affecting SHBG

• Elevated SHBG (>50 nmol/L) occurs in: aging, hyperthyroidism, hepatic disease, HIV infection, and creates functional hypogonadism with normal total testosterone 3, 8
• Decreased SHBG occurs in: obesity, metabolic syndrome, hypothyroidism, and can mask true hypogonadism by maintaining normal total testosterone 1, 5

Diagnostic Algorithm for Hypogonadism

Step 1: Initial Screening

• Measure morning total testosterone (8-10 AM, fasting) on two separate occasions to confirm persistent low levels 1, 5, 4
• If total testosterone is <150 ng/dL: hypogonadism is confirmed with 98.9% specificity—proceed to treatment evaluation 2
• If total testosterone is 150-280 ng/dL: likely hypogonadism but requires free testosterone confirmation 2
• If total testosterone is 280-400 ng/dL: mandatory free testosterone measurement, as 17.2% will have normal total but low free testosterone 2, 3
• If total testosterone is >400 ng/dL: hypogonadism is effectively excluded in most cases 2

Step 2: Free Testosterone Assessment (When Indicated)

• Measure free testosterone by equilibrium dialysis (gold standard) or calculate using the Vermeulen equation with total testosterone, SHBG, and albumin 1, 6, 7
• Measure SHBG simultaneously to understand the relationship between total and free testosterone 1, 5
• Free testosterone <6.5 ng/dL confirms hypogonadism regardless of total testosterone level 3, 8

Step 3: Distinguish Primary vs Secondary Hypogonadism

• Measure LH and FSH after confirming low testosterone to determine the etiology 1, 5
• Elevated LH/FSH with low testosterone = primary (testicular) hypogonadism 1
• Low or low-normal LH/FSH with low testosterone = secondary (pituitary-hypothalamic) hypogonadism 1
• This distinction is critical: men with secondary hypogonadism desiring fertility require gonadotropin therapy (hCG + FSH), not testosterone replacement 1

Performance Characteristics of Total Testosterone

Sensitivity Analysis

• At <280 ng/dL threshold: 91.0% sensitivity—misses 9% of men with low free testosterone 2
• At <350 ng/dL threshold: 96.8% sensitivity—misses 3.2% of men with low free testosterone 2
• At <400 ng/dL threshold: 98.2% sensitivity—misses only 1.8% of men with low free testosterone 2

Specificity Analysis

• At <280 ng/dL threshold: 73.7% specificity—26.3% false positive rate 2
• At <200 ng/dL threshold: 92.6% specificity—7.4% false positive rate 2
• At <150 ng/dL threshold: 98.9% specificity—only 1.1% false positive rate 2

The key clinical implication: total testosterone between 280-400 ng/dL is neither sensitive enough to exclude hypogonadism nor specific enough to confirm it—free testosterone measurement is mandatory in this range 2, 3

Clinical Correlations: Free vs Total Testosterone

Parameters That Correlate Better with Free Testosterone

• Age, hematocrit, gonadotropins, gynecomastia, BMI, number of comorbidities, erectile dysfunction, and low libido all correlate significantly with free testosterone 8
• Total testosterone correlates only with BMI and low libido—missing most clinical manifestations of androgen deficiency 8

Diagnostic Accuracy in Sexual Dysfunction

• In men presenting with erectile dysfunction, 8.4% have hypogonadism that is misdiagnosed by total testosterone alone 8
• In men with infertility, 2% have hypogonadism missed by total testosterone screening 8
• Free testosterone assessment allows more accurate diagnosis of functional hypogonadism, especially in men with sexual symptoms 8

Common Pitfalls to Avoid

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

• Never screen men over 60 years with total testosterone alone—26.3% will have normal total but low free testosterone 3
• Never diagnose or exclude hypogonadism in obese men without free testosterone measurement—SHBG alterations create significant discordance 1, 5
• Never use total testosterone as the sole criterion when levels are 280-400 ng/dL—this range has inadequate sensitivity and specificity 2

Do Not Use Free Androgen Index as a Substitute

• Free androgen index (total testosterone/SHBG × 100) correlates poorly with calculated free testosterone (r² = 0.21-0.46) 7
• Free androgen index overestimates free testosterone at low SHBG concentrations, leading to missed diagnoses 7

Do Not Diagnose Hypogonadism on a Single Measurement

• Testosterone exhibits significant diurnal variation and measurement variability—always confirm with a second morning measurement 5, 4
• Biologic factors cause substantial variation over time—single measurements are insufficient for diagnosis 4

Treatment Implications

When to Initiate Testosterone Therapy

• Testosterone therapy should only be initiated when both biochemical hypogonadism is confirmed (low free testosterone on two separate measurements) AND specific symptoms are present, particularly diminished libido and erectile dysfunction 9, 1
• The American College of Physicians recommends discussing potential benefits, harms, costs, and patient preferences before initiating treatment 9
• Expected benefits are modest: small but significant improvements in sexual function (standardized mean difference 0.35), with little to no effect on physical functioning, energy, vitality, or cognition 9, 1

Monitoring During Treatment

• Recheck testosterone levels 2-3 months after initiating therapy or any dose adjustment, targeting mid-normal levels (500-600 ng/dL) 1, 5
• Monitor hematocrit periodically and withhold treatment if >54%—injectable testosterone carries higher erythrocytosis risk 1
• Reevaluate symptoms at 12 months and discontinue testosterone if no improvement in sexual function 9, 1