Essential Laboratory Testing for Free Testosterone Assessment
The most critical blood test for determining free testosterone is a morning (8-10 AM) serum free testosterone measured by equilibrium dialysis, accompanied by total testosterone, sex hormone-binding globulin (SHBG), and albumin levels—all drawn on two separate mornings to confirm the diagnosis. 1
Primary Blood Tests Required
Core Testosterone Panel
- Morning total testosterone (8-10 AM): Must be drawn between 8-10 AM due to diurnal variation, with levels highest in early morning; normal range is 300-800 ng/dL in most laboratories 1
- Free testosterone by equilibrium dialysis: This is the gold standard method and the most accurate measure of metabolically active testosterone 1, 2
- Sex hormone-binding globulin (SHBG): Essential because obesity and other conditions alter SHBG levels, which can cause low total testosterone with normal free testosterone 1
- Albumin: Required for accurate calculation of free testosterone when equilibrium dialysis is unavailable 1
Critical timing requirement: Both total and free testosterone must be measured on at least two separate occasions in early morning (8-10 AM) to confirm the diagnosis, as testosterone levels show significant diurnal variation and assay variability 1, 2
Calculated Free Testosterone Alternative
If equilibrium dialysis is unavailable, calculated free testosterone using validated formulas incorporating total testosterone, SHBG, and albumin is acceptable for most clinical situations 1, 2, 3. However, accuracy decreases significantly when albumin is ≤3.5 g/dL combined with SHBG ≤30 nmol/L, occurring in approximately 1.2% of cases—in these situations, equilibrium dialysis measurement is mandatory 4.
Essential Follow-Up Tests When Testosterone is Low
Pituitary Hormone Assessment
- Luteinizing hormone (LH): Must be measured to distinguish primary (testicular) from secondary (pituitary-hypothalamic) hypogonadism 1, 2
- Follicle-stimulating hormone (FSH): Required alongside LH to complete the hypogonadism workup 1
Interpretation algorithm: Low testosterone with low or low-normal LH/FSH indicates secondary hypogonadism and requires additional pituitary evaluation 1, 2
Additional Endocrine Testing for Secondary Hypogonadism
When LH/FSH are low or low-normal with confirmed low testosterone:
- Serum prolactin: Screen for hyperprolactinemia as a cause of secondary hypogonadism 1, 2
- Iron saturation: Evaluate for hemochromatosis affecting pituitary function 1
- Pituitary MRI: Required for men with testosterone <150 ng/dL combined with low or low-normal LH, regardless of prolactin levels 2
Estradiol Assessment
- Serum estradiol: Measure in patients presenting with gynecomastia or breast symptoms, as increased aromatization of testosterone to estradiol in adipose tissue can suppress pituitary LH secretion 1, 2
Pre-Treatment Safety Testing
Before initiating testosterone replacement therapy:
- Hemoglobin/hematocrit: Baseline measurement required as testosterone stimulates erythropoiesis 2, 5
- Cardiovascular risk assessment: Document cardiovascular disease risk factors 2
Important Factors Affecting Testosterone Levels
Obesity-Related Changes
In obese men, low total testosterone is frequently due to low SHBG concentrations, while free testosterone remains normal—this is why measuring free testosterone is essential in obesity 1. A subset of obese men will have frankly low free testosterone due to increased aromatization of testosterone to estradiol in adipose tissue, causing estradiol-mediated negative feedback on pituitary LH secretion 1.
Binding Protein Alterations
- SHBG variations: Conditions altering SHBG (obesity decreases it, liver disease and hyperthyroidism increase it) significantly affect the total testosterone-to-free testosterone relationship 1, 3
- Albumin levels: Low albumin (<3.5 g/dL) combined with low SHBG (<30 nmol/L) creates significant variance in calculated free testosterone, requiring direct measurement 4
Critical Diagnostic Thresholds
- Total testosterone <300 ng/dL: Reasonable cut-off supporting low testosterone diagnosis 1
- Total testosterone 280-350 ng/dL: Not sensitive enough to reliably exclude hypogonadism; free testosterone measurement is mandatory in this range 6
- Total testosterone >350-400 ng/dL: Reliably predicts normal free testosterone 6
- Total testosterone <150 ng/dL: Highly specific for hypogonadism and warrants pituitary MRI if LH is low or low-normal 2, 6
Common Pitfalls to Avoid
Do not use screening questionnaires as substitutes for laboratory testosterone measurement—they have variable specificity and sensitivity and are not appropriate for identifying candidates for testosterone therapy 1, 2.
Do not rely on free androgen index (total testosterone/SHBG ratio) in men, as it is inaccurate at extremes of SHBG concentration 1, 7, 8.
Do not use analog immunoassay methods for free testosterone measurement available at most local laboratories—these have limited reliability compared to equilibrium dialysis 1, 3.
Do not measure testosterone at random times—afternoon or evening measurements will miss the diagnosis due to diurnal variation 1.
Urine and Saliva Testing
No urine or saliva tests are recommended in current clinical practice guidelines for testosterone assessment 1. Approximately 90% of testosterone is excreted in urine as glucuronic and sulfuric acid conjugates, but this is not used diagnostically 5, 9.