EndoAxis Clinical Team
Hormone testing is central to personalized and functional medicine, giving clinicians a window into the signals that regulate metabolism, mood, fertility, vitality, and aging. But with multiple testing methods—and hormone levels fluctuating across the day, month, and lifespan—choosing when and how to test is just as important as choosing what to test.This guide summarizes the key physiology behind hormone rhythms, outlines evidence-based testing methods, and clarifies ideal timing for testing in women, men, and patients using hormone therapy.
Steroid Hormone Basics
Steroid hormones—estrogens, progesterone, testosterone, DHEA, cortisol, aldosterone, and vitamin D—are cholesterol-derived and extremely potent. Their levels shift throughout the day and, in cycling women, across the menstrual month. Because these hormones are produced in tiny quantities, testing methodologies must be sensitive and specific enough to measure very small concentrations accurately.
Hormone Physiology & Expected Ranges
Cycling Women
Estradiol (E2)
Produced daily by granulosa cells, estradiol fluctuates across the cycle:
- Follicular phase: 19–144 pg/mL (ideal 50–80)
- Ovulation surge: 64–357 pg/mL (ideal >150)
- Luteal phase: 56–214 pg/mL (ideal 120–180)
- Postmenopausal: <50 pg/mL
Estradiol rises sharply before ovulation and remains steady in the luteal phase to support endometrial stability.
Progesterone
Progesterone rises only after ovulation through corpus luteum production; adrenal output is minimal.
Luteal phase: 5–21.5 ng/mL
• >5 ng/mL indicates ovulation
• >10 ng/mL supports fertility
• >15 ng/mL is ideal
Follicular/Postmenopausal: <1 ng/mL
Test only during the luteal phase, ideally 5–7 days after ovulation.
Androgens (Testosterone & Androstenedione)
Women produce small amounts via the ovaries, with most of their androgen activity occurring in peripheral conversion from androstenedione.
Total testosterone: 2–45 ng/dL (ideal 20–45)
Like men, women exhibit a morning peak, so testing is best before 11 a.m.
Men
Testosterone
Produced by Leydig cells and strongly diurnal.
Test in the morning before 11 am per guidelines (ideally 6–10 a.m.), fasting.
Two non-sequential tests are recommended for accuracy.
Ranges:
- Total testosterone: 300–1100 ng/dL (functional ideal 500–900 ng/dL)
- Free testosterone: ideal = 150–225 pg/mL (preferably measured via equilibrium dialysis or calculated with Vermeulen equation)
DHT (5α-dihydrotestosterone)
More potent than testosterone and primarily produced in the prostate and skin.
- DHT: 12–65 ng/dL (optimal 40–65)
Estradiol in Men
Supports sperm formation and is produced via aromatization of testosterone. Most circulating estrogens in men come from peripheral conversion.
- Estradiol: 1–50 pg/mL (ideal 25–40, depending on T/E ratio)
Progesterone in Men
Assists sperm function, Inhibin regulation, and 5α-reductase inhibition.
- Progesterone: 0.2–1.3 ng/mL (optimal 0.35–1.0 ng/mL)
Adrenal Hormones
Cortisol
- Follows a strong diurnal rhythm with a morning peak.
- Best test: 4-point salivary curve
- Serum: fasting, early morning only
- Measured in mcg/dL
DHEA-S
The circulating fraction of DHEA is DHEA-s. DHEA-s is produced only in the adrenal cortex via SULT2A1.
- Levels peak in the 20s; declines 1–5% annually after age 30.
- Measured in mcg/dL.
Why Testing Methodology Matters
Steroid hormones are tiny in terms of circulating amounts, and are measured across dramatically different concentration scales. For example:
Because estradiol, testosterone and free testosterone circulate in tiny quantities, poor testing methods can easily miss or misrepresent them.
As we are trying to capture such small fractions of hormone, we need to understand what lab test methods exist, and what they mean in terms of accuracy.
First, we need to define what it means to be sensitive and specific, as it relates to lab validity.
Core Analytical Principles for Hormone Testing
Sensitivity
- Ability to detect true positives.
- High sensitivity → negative test rules OUT.
- (S-N-OUT)
Specificity
- Ability to detect true negatives.
- High specificity → positive test rules IN.
- (S-P-IN)
Accuracy
- Closeness to the true value.
Precision
- Reproducibility of results.
- (A test can be precise but inaccurate, and vice versa.)
Sensitivity and Specificity relate to the methodology used for analyte evaluation.
Accuracy and Precision relate to analyte capture, processing, lab handling, instrumentation and calibration.
A lab test ideally has both high accuracy and high precision, indicating both the methodology and instrumentation are well-calibrated and stable.
Optimal testing methods (e.g., LC–MS/MS) maximize both accuracy and precision.
Testing Technologies: What Clinicians Should Know
Immunoassay (IA)
- Pros: Fast, inexpensive
- Cons: Low specificity, poor sensitivity for low-concentration hormones (E2, T, P)
LC–MS/MS
- Pros: Highest clinical accuracy for sex steroids; best for low-level detection;
- minimal cross-reactivity
- Cons: Higher cost
- Recommended method for most sex hormone testing.
GC–MS
- Pros: Extremely specific; ideal for reference confirmation
- Cons: Slow; not routinely used clinically
Equilibrium Dialysis
- Gold standard for free testosterone or free serum cortisol
- Labor-intensive, but most accurate. Not readily available.
Best Practice Timing Recommendations
General
- All sex hormones: Test in the morning before 10 a.m.
- Hormones are pulsatile—progesterone can shift up to 80% within 90 minutes during the luteal phase, while testosterone will peak in the morning, with a natural, expected and gradual decline throughout the day.
Cycling Women
- Luteal phase (Day ~19–22 of a 28-day cycle): Estradiol, progesterone, Inhibin A
- Follicular phase (Days 2–5): FSH, LH, AMH, prolactin, Inhibin B, 17-OH progesterone
Postmenopausal Women
- No specific cycle timing; morning testing preferred.
- LC–MS/MS recommended for low-level estradiol and testosterone.
Men
- Test testosterone 6–10 a.m., fasting, twice on separate days.
Key Takeaways
- Hormones vary by time of day, menstrual phase, and age—timing dramatically affects interpretation.
- LC–MS/MS provides the most reliable results for sex steroids.
- Cycling women require phase-specific testing; men require morning, repeat testosterone evaluation.
- Understanding sensitivity, specificity, accuracy, and precision helps clinicians choose appropriate methodologies and avoid misleading results.
