What is the proper procedure for testing serum testosterone levels in adult males?

How to Test Serum Testosterone

Measure morning serum total testosterone between 8 AM and 10 AM on at least two separate days using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a laboratory certified by the CDC's Hormone Standardization Program, and confirm levels below 300 ng/dL before diagnosing hypogonadism. 1, 2, 3

Timing of Blood Draw

• Collect blood samples between 8:00 AM and 10:00 AM to capture peak testosterone levels, as this timing is critical for accurate diagnosis in men of all ages 2, 3, 4
• Morning testosterone levels can be 20-25% higher than afternoon levels in younger men (ages 30-40), with this difference declining to approximately 10% by age 70 4
• While some evidence suggests afternoon testing may be acceptable in men ≥45 years for total testosterone, morning collection remains the gold standard to avoid missing cases of hypogonadism 5, 6
• The diurnal variation affects free and bioavailable testosterone more significantly than total testosterone, making morning draws especially important when measuring these parameters 5

Required Testing Protocol

• Obtain fasting samples on at least two separate mornings to confirm persistently low testosterone, as single measurements are insufficient due to assay variability and biological fluctuation 1, 3, 7
• Measure total testosterone first as the primary screening test, with levels <300 ng/dL indicating potential hypogonadism 1, 2
• If total testosterone is confirmed low on repeat testing, measure luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to distinguish primary testicular failure (elevated LH/FSH) from secondary hypothalamic-pituitary dysfunction (low or low-normal LH/FSH) 1, 2

Laboratory Method Selection

• Use liquid chromatography-tandem mass spectrometry (LC-MS/MS) rather than immunoassays, as this method provides superior accuracy and precision 3, 7
• Ensure the laboratory is certified by the CDC's Hormone Standardization (HoST) Program to allow application of harmonized reference ranges 3, 7
• Immunoassays are less reliable, particularly at low testosterone concentrations, and should be avoided when diagnostic accuracy is critical 3, 7

Additional Hormone Measurements

Free Testosterone Testing

• Measure free testosterone by equilibrium dialysis when total testosterone is borderline (near 300 ng/dL) or when alterations in binding proteins are suspected 1, 2, 3, 7
• Free testosterone is particularly important in men with obesity, as increased sex hormone-binding globulin (SHBG) can affect total testosterone interpretation 2
• Calculate the free androgen index (total testosterone ÷ SHBG) as an alternative when equilibrium dialysis is unavailable, though this is less reliable than direct measurement 1
• Avoid analog free testosterone assays available at most local laboratories, as these have limited reliability 1

Sex Hormone-Binding Globulin (SHBG)

• Measure SHBG levels in conjunction with total testosterone, especially in men with obesity or metabolic syndrome where SHBG may be altered 2
• SHBG measurement helps distinguish true hypogonadism from low SHBG-related decreases in total testosterone 2

Secondary Hypogonadism Workup

• Measure serum prolactin in patients with low total testosterone and low or low-normal LH levels to screen for hyperprolactinemia 1
• If prolactin is elevated, repeat the measurement to ensure the initial elevation was not spurious 1
• Men with total testosterone <150 ng/dL combined with low or low-normal LH should undergo pituitary MRI regardless of prolactin levels, as non-secreting adenomas may be present 1

Pre-Treatment Baseline Testing

Before initiating testosterone therapy, obtain:

• Hemoglobin/hematocrit to establish baseline and identify pre-existing polycythemia (withhold therapy if hematocrit >50%) 1
• Prostate-specific antigen (PSA) in men over 40 years to exclude occult prostate cancer 1
• Digital rectal examination to assess for palpable prostate abnormalities 1
• Lipid panel and cardiovascular risk assessment including blood pressure, diabetes screening, and smoking status 1

Common Pitfalls to Avoid

• Never diagnose hypogonadism based on a single testosterone measurement, as biological and assay variability can produce spurious results 1, 3, 7
• Never test testosterone in the afternoon or evening in men under 75 years, as this can miss 17-20% of hypogonadal cases due to diurnal variation 4
• Never use symptoms alone to diagnose hypogonadism without biochemical confirmation, as symptoms lack specificity 1, 2
• Never skip LH/FSH measurement after confirming low testosterone, as distinguishing primary from secondary hypogonadism is critical for treatment selection and fertility preservation 1, 2
• Never initiate testosterone therapy without confirming the patient does not desire fertility, as exogenous testosterone suppresses spermatogenesis and causes azoospermia 1, 2, 8

Monitoring During Treatment

• Measure testosterone levels 2-3 months after treatment initiation and after any dose change 2
• For injectable testosterone (cypionate/enanthate), measure levels midway between injections (days 5-7), targeting mid-normal values of 500-600 ng/dL 2
• Once stable levels are achieved, monitor every 6-12 months with testosterone, hematocrit, and PSA measurements 1, 2
• Withhold treatment if hematocrit exceeds 54% and consider phlebotomy in high-risk cases 1, 2