Laboratory Testing for Testosterone
Measure morning total testosterone (before 11:00 AM) using a reliable assay, and confirm the diagnosis with a second morning measurement if the initial value is low or equivocal. 1, 2
Initial Testing Approach
- Obtain morning total testosterone as the primary diagnostic test, collected before 11:00 AM to account for diurnal variation in testosterone production 1, 3
- Use an accurate and reliable assay, ideally liquid chromatography tandem mass spectrometry (LC-MS/MS), in a laboratory certified by an accuracy-based benchmark 4
- Confirm any low result with a second morning measurement, as a single low reading is insufficient for diagnosis due to significant intra-individual variability 1, 5, 2
Diagnostic Thresholds
- Low testosterone is defined as consistently <300 ng/dL (<9.0 nmol/L) on at least two early morning measurements 1
- Values between 7.0-12.5 nmol/L (approximately 200-360 ng/dL) are considered equivocal and require repeat testing 3
- Diagnosis requires BOTH low testosterone measurements AND clinical symptoms/signs of deficiency 1
Additional Hormone Testing
When testosterone is confirmed low, obtain the following to establish etiology:
- Serum luteinizing hormone (LH) to determine if hypogonadism is primary (testicular) or secondary (hypothalamic-pituitary) 6, 5
- Serum follicle-stimulating hormone (FSH) as part of the complete gonadal axis assessment 6, 7
- Serum prolactin in patients with low testosterone and low/normal LH levels to screen for hyperprolactinemia 6, 5
- Sex hormone-binding globulin (SHBG) for routine assessment 7
Free or Bioavailable Testosterone
- Measure free or bioavailable testosterone when total testosterone is near the lower limit of normal (around 9.0 nmol/L or 260 ng/dL) 3
- Also measure in patients with suspected alterations in binding protein concentrations (obesity, aging, liver disease, thyroid disorders) 4
- Free testosterone can be measured directly (equilibrium dialysis method) or calculated using mathematical expressions incorporating total testosterone, SHBG, and albumin 3, 7
Important Testing Considerations
- Fasting state is preferred for optimal accuracy 4
- Be aware that laboratory reference ranges vary widely (low values ranging from 130-450 ng/dL across different labs), representing a 350% difference 8
- Automated immunoassays are commonly used but may have quality issues; LC-MS/MS provides more specific estimates 3, 4
- Testosterone levels fluctuate seasonally and day-to-day, supporting the need for repeat measurements in equivocal cases 7
Imaging and Further Workup
- Consider pituitary MRI if total testosterone is low with low/normal LH levels, as non-secreting adenomas may be present 6, 5
- This is particularly important when testosterone is <150 ng/dL with inappropriately normal gonadotropins 5
Common Pitfalls to Avoid
- Do not diagnose testosterone deficiency based on a single measurement 1, 2
- Do not test testosterone in the afternoon or evening due to diurnal variation 1, 3
- Do not rely solely on total testosterone when values are borderline; obtain free or bioavailable testosterone 3, 4
- Do not diagnose hypogonadism without corresponding clinical symptoms, regardless of laboratory values 1, 2