Which test should be performed to evaluate testosterone levels?

Testosterone Testing Protocol

Measure serum total testosterone using two separate early morning (before 10-11 AM) blood samples, with both values below 300 ng/dL required to diagnose testosterone deficiency. 1

Testing Methodology

Initial Test: Total Testosterone

• Timing: Fasting morning sample (before 11:00 AM) 1, 2
• Method: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is preferred over automated immunoassays for accuracy 3
• Laboratory: Use a CDC-certified laboratory with the same method/instrumentation for both measurements 1
• Diagnostic threshold: Total testosterone <300 ng/dL (10.4 nmol/L) 1

Confirmation Requirement

You must obtain a second morning measurement on a separate day because:

• 30% of men with initial low-normal values will have normal levels on repeat testing 2
• 15% of healthy young men may have a single low value in a 24-hour period 2
• Day-to-day variations are substantial enough that single measurements are inadequate 2

When to Measure Free or Bioavailable Testosterone

Add free testosterone testing when:

• Total testosterone is 280-350 ng/dL (borderline low) 1, 2
• Conditions affecting SHBG are present:
  • Obesity
  • Diabetes mellitus
  • Aging (>65 years)
  • Thyroid disease
  • Chronic illness 2

Free Testosterone Measurement Options

• Gold standard: Equilibrium dialysis method (reference range 66-309 pg/mL in all men; 120-368 pg/mL in men 19-39 years) 4
• Alternative: Calculate from total testosterone, SHBG, and albumin 2
• Avoid: Analog immunoassays for free testosterone—these are inaccurate and affected by SHBG alterations 2

Adjunctive Testing

Measure serum luteinizing hormone (LH) in all patients with low testosterone to differentiate primary from secondary hypogonadism 1

Critical Pitfalls to Avoid

1. Do not use afternoon samples—the circadian rhythm causes lower afternoon values, though this is blunted in older men 2
2. Do not rely on questionnaires alone—they have poor sensitivity/specificity and should not replace laboratory testing 1
3. Do not use saliva testing for monitoring transdermal testosterone therapy—saliva values become supraphysiologic and do not correlate with clinical response 5
4. Do not diagnose TD on testosterone levels alone—diagnosis requires both low testosterone AND clinical symptoms/signs 1

Clinical Context for Testing

Consider testosterone measurement even without symptoms in men with:

• Unexplained anemia
• Bone density loss
• Diabetes
• HIV/AIDS
• Chronic narcotic use
• Male infertility
• Pituitary dysfunction
• History of chemotherapy or testicular radiation
• Chronic corticosteroid use 1

The 2018 AUA guideline provides the strongest evidence base (Grade A for the two-measurement requirement), superseding the 2010 Endocrine Society guideline which established similar principles but with slightly different thresholds.