Optimal Timing for Testosterone Level Testing in Women on TRT
For women on testosterone replacement therapy, testosterone levels should be measured in the morning (before 10 AM) in a fasting state, ideally midway through the dosing interval for injectable formulations or at any time after 2-3 months of stable therapy for transdermal preparations. 1
Critical Guideline Context
Before proceeding with monitoring, it's essential to recognize that major medical societies explicitly recommend against routine testosterone testing in women in nonresearch settings (strong recommendation, low quality evidence). 2 The Infectious Diseases Society of America specifically reserves morning testosterone testing for adult men with symptoms like decreased libido or erectile dysfunction, not for women. 2
However, when testosterone therapy has been initiated in women (typically off-label), proper monitoring becomes necessary despite limited guideline support.
Timing Based on Formulation Type
For Injectable Testosterone
- Measure testosterone levels midway between injections, targeting mid-normal values (500-600 ng/dL for male reference ranges, though female target ranges are not well-established). 1
- Peak serum levels occur 2-5 days after intramuscular injection, with return to baseline by days 10-14. 1
- Avoid testing at peak (days 2-5) as this shows supraphysiologic levels that don't reflect average exposure and may lead to inappropriate dose reduction. 3
- Avoid testing at trough (days 13-14) as levels may have returned to baseline, potentially leading to unnecessary dose escalation. 3
For Transdermal Testosterone (Gel, Patch, Cream)
- Levels can be measured at any time after 2-3 months of stable therapy, as transdermal preparations provide more stable day-to-day testosterone levels. 3, 4
- Morning measurement (before 10 AM) remains preferred to maintain consistency with baseline testing. 5, 6
Fasting State Considerations
Women should be tested in a fasting state, as research demonstrates that fasting testosterone levels are significantly higher than non-fasting levels in apparently healthy women of childbearing age (mean 27.39 ng/dL fasting vs 24.47 ng/dL non-fasting, p=0.01). 6 This difference ensures accurate assessment and consistency across serial measurements.
Initial Monitoring Schedule
- First measurement: 2-3 months after treatment initiation or after any dose change. 1
- First follow-up visit: 1-2 months to assess clinical response and consider dose escalation if symptoms persist with suboptimal levels. 1
- Subsequent monitoring: Every 3-6 months for the first year, then every 6-12 months once stable levels are confirmed. 7, 1
Essential Concurrent Measurements
Beyond testosterone measurement alone:
- Measure SHBG alongside testosterone to calculate the free testosterone index (total testosterone/SHBG ratio), as this provides better assessment of androgen status than total testosterone alone. 2
- Free testosterone by equilibrium dialysis should be obtained when total testosterone is borderline or when SHBG alterations are suspected. 5
- Monitor hematocrit/hemoglobin at each visit, as testosterone therapy carries risk of erythrocytosis—withhold treatment if hematocrit exceeds 54%. 3
Critical Pitfalls to Avoid
- Never rely on a single testosterone measurement due to substantial biologic and measurement variation over time. 5
- Never test during peak levels (days 2-5 post-injection) as this misrepresents average testosterone exposure. 3
- Never skip SHBG measurement, as elevated SHBG can create discordance between total and free testosterone, masking true androgen status. 2
- Never assume non-fasting samples are equivalent to fasting samples in women, as this introduces systematic measurement error. 6
Assay Considerations
Total testosterone should ideally be measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) in a laboratory certified by an accuracy-based benchmark, as this provides superior accuracy compared to immunoassays, particularly at the lower testosterone concentrations typical in women. 5