How to Test Your Testosterone Levels
How to Test Your Testosterone Levels
The Complete Clinical Guide to Accurate Hormone Testing
KEY TAKEAWAYS
- Morning testing (7-10 AM) is essential for accurate results—testosterone follows a circadian rhythm with peak levels in early morning
- The Endocrine Society requires TWO separate morning tests on different days to confirm low testosterone
- LC-MS/MS (liquid chromatography-mass spectrometry) is the gold standard testing method for accuracy
- A complete hormone panel (LH, FSH, SHBG, prolactin) helps differentiate primary from secondary hypogonadism
- Diagnosis requires BOTH laboratory confirmation AND clinical symptoms—a number alone doesn't warrant treatment
Why Accurate Testosterone Testing Matters
Testosterone is far more than just a "male hormone." It orchestrates virtually every aspect of men's physical and mental health—from muscle mass and bone density to mood stability, cognitive function, and sexual performance. When levels decline, the effects ripple through every system in your body, yet many men suffer for years without understanding the root cause of their symptoms.
The challenge is that accurately measuring testosterone requires more than simply drawing blood. Testosterone levels fluctuate dramatically throughout the day, respond to food intake, vary based on sleep quality, and can be artificially suppressed by medications, illness, and stress. Without proper testing methodology, you may receive results that don't reflect your true hormonal status—potentially leading to either unnecessary treatment or missed diagnosis.
This comprehensive guide covers everything you need to know about testosterone testing: the science behind different measurement methods, how to prepare for accurate results, what your numbers actually mean, and what additional tests may be necessary for a complete clinical picture.
When Should You Get Your Testosterone Tested?
The Endocrine Society's 2018 Clinical Practice Guidelines explicitly recommend against routine testosterone screening in the general population.1 However, testing is clinically indicated when you experience symptoms suggestive of testosterone deficiency combined with risk factors that increase likelihood of hypogonadism.
Symptoms That Warrant Testing
Sexual and Reproductive Symptoms
- Decreased libido (reduced sex drive)
- Erectile dysfunction or difficulty maintaining erections
- Reduced spontaneous morning erections
- Infertility or reduced sperm production
- Gynecomastia (breast tissue enlargement)
Physical Symptoms
- Unexplained fatigue and decreased energy
- Loss of muscle mass despite regular exercise
- Increased body fat, particularly abdominal obesity
- Decreased bone mineral density or osteoporosis
- Loss of body hair (armpit, pubic, facial)
- Hot flashes or night sweats
Psychological Symptoms
- Depression or depressed mood
- Irritability and mood changes
- Difficulty concentrating or "brain fog"
- Decreased motivation and sense of well-being
High-Risk Populations
Certain conditions significantly increase the risk of testosterone deficiency and warrant proactive testing:
- Type 2 diabetes mellitus (30-50% have low testosterone)
- Obesity (BMI >30), particularly morbid obesity (BMI >40)
- Chronic opioid therapy (any duration >90 days)
- HIV/AIDS infection
- End-stage renal disease or chronic kidney disease
- History of chemotherapy or radiation therapy
- Pituitary disorders or head trauma
- Chronic glucocorticoid (steroid) use
- Sleep apnea (both a cause and consequence)
- Unexplained anemia or low bone mineral density
Understanding the Different Types of Testosterone Tests
Testosterone circulates in your blood in three distinct forms, each with different clinical significance. Understanding these fractions is essential for accurate interpretation of your results.
The Three Fractions of Circulating Testosterone
| Fraction | Percentage | Clinical Significance |
|---|---|---|
| Free Testosterone | 1-3% of total | Biologically active; directly available to tissues |
| Albumin-Bound | 35-50% of total | Weakly bound; can dissociate and become available |
| SHBG-Bound | 50-60% of total | Tightly bound; essentially inactive |
Total Testosterone
Total testosterone measures the combined amount of all three fractions circulating in your blood. It is the initial test of choice recommended by the Endocrine Society and American Urological Association for diagnosing testosterone deficiency.1,2
The American Urological Association defines low testosterone as a total testosterone level below 300 ng/dL, while the Endocrine Society's CDC-harmonized reference establishes the lower limit at 264 ng/dL.1,2 However, these cutoffs represent population-based thresholds rather than individualized targets, and symptoms often correlate more strongly with free testosterone levels.
Free Testosterone
Free testosterone represents the unbound fraction that is immediately available to enter cells and activate androgen receptors. While comprising only 1-3% of total testosterone, it is often the most clinically relevant measure—particularly when SHBG levels are abnormal.
When is free testosterone testing indicated? The Endocrine Society recommends measuring free testosterone when total testosterone is near the lower limit of normal (264-400 ng/dL) or when conditions that alter SHBG are present:1
Conditions that DECREASE SHBG (may falsely lower total T):
- Obesity
- Type 2 diabetes mellitus
- Nephrotic syndrome
- Hypothyroidism
- Glucocorticoid and androgen use
Conditions that INCREASE SHBG (may mask low free T):
- Aging
- Hyperthyroidism
- Liver cirrhosis
- HIV infection
- Anticonvulsant medications
- Estrogen therapy
Bioavailable Testosterone
Bioavailable testosterone combines free testosterone with the albumin-bound fraction (since albumin binding is weak and reversible). This represents 40-50% of total testosterone and may better reflect tissue availability than either total or free testosterone alone.
The Vermeulen formula, developed by Dr. Alex Vermeulen and validated against equilibrium dialysis (the reference standard), provides accurate calculated estimates of free and bioavailable testosterone from total testosterone, SHBG, and albumin measurements.3
Testing Methodology: Not All Tests Are Created Equal
The accuracy of your testosterone results depends heavily on the laboratory method used. Understanding the difference between testing methodologies can help you ensure you receive clinically meaningful results.
LC-MS/MS: The Gold Standard
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recognized by the Endocrine Society and CDC as the most accurate method for testosterone measurement.4 This technology actually identifies the testosterone molecule by its chemical structure, eliminating interference from structurally similar compounds.
LC-MS/MS advantages:
- Superior accuracy across all concentration ranges
- Particularly reliable at low concentrations (<100 ng/dL)
- Minimal interference from other steroids
- Traceable to CDC reference standards
- Inter-laboratory harmonization achievable (<5% variance)
Immunoassays: Widely Available but Variable
Immunoassays use antibodies to detect testosterone and are widely available in most clinical laboratories. However, significant limitations exist:
- Cross-reactivity with structurally similar steroids
- Reduced accuracy at low concentrations (can vary up to 20% from LC-MS/MS)5
- Significant inter-laboratory variability
- Different manufacturers may yield different results on the same sample
The CDC's Hormone Standardization (HoSt) Program certifies testosterone assays that meet accuracy standards. If using an immunoassay, verify that the laboratory uses a CDC-certified method.4
Direct Free Testosterone Assays: Not Recommended
The Endocrine Society explicitly recommends against using direct analog-based free testosterone immunoassays because they are inaccurate.1 These tests use a labeled testosterone analog that doesn't accurately reflect the equilibrium between bound and free testosterone. If free testosterone measurement is needed, calculate it from total testosterone and SHBG using validated formulas (Vermeulen equation) or use equilibrium dialysis.
How to Prepare for Accurate Testosterone Testing
Proper preparation is critical for obtaining accurate, clinically meaningful results. Multiple factors can temporarily suppress or elevate testosterone levels, potentially leading to misdiagnosis.
Timing: The Morning Window Is Non-Negotiable
Testosterone follows a circadian rhythm, with levels peaking in early morning and declining throughout the day. The Endocrine Society mandates testing between 7:00 AM and 10:00 AM for accurate assessment.1
This diurnal variation is most pronounced in younger men (under 45). Studies show that a substantial fraction of men with afternoon testosterone levels in the hypogonadal range will have normal concentrations when measured in the morning.1 Testing later in the day can result in falsely low readings and inappropriate diagnosis.
Night shift workers: If you consistently work nights, discuss testing timing with your provider. Testing should occur within 3 hours of your normal wake time, after your sleep-wake cycle has stabilized (not during shift changes or jet lag).6
Fasting: Recommended but Evolving Evidence
The Endocrine Society recommends fasting for testosterone measurement because food intake can acutely suppress testosterone levels.1 Glucose and insulin spikes following meals can transiently reduce testosterone by 25% or more.
Fasting guidelines:
- Fast for 8-12 hours before your test (water is permitted)
- Avoid caloric beverages (juice, coffee with cream/sugar)
- Schedule your test early morning to minimize fasting duration
- Take morning medications with water unless instructed otherwise
Medications and Substances That Affect Results
Several medications can significantly suppress testosterone levels. Inform your provider about all medications before testing:
Medications that suppress testosterone:
- Opioids: Even short-term use suppresses the hypothalamic-pituitary axis; men on fentanyl have 25x higher odds of androgen deficiency vs. hydrocodone7
- Glucocorticoids: Chronic prednisone/steroid use suppresses the HPT axis
- Ketoconazole: Directly inhibits testosterone synthesis
- GnRH agonists/antagonists: Used in prostate cancer treatment
- Spironolactone: Anti-androgen effects
- Finasteride/dutasteride: May slightly affect testosterone metabolism
Other factors that can suppress results:
- Acute illness: Any systemic illness temporarily suppresses testosterone—wait 4+ weeks after recovery
- Sleep deprivation: 5 days of 5-hour sleep reduces testosterone by 15%8
- Alcohol: Heavy acute intake can decrease testosterone by 23%
- Strenuous exercise: Avoid intense exercise 24-48 hours before testing
- High-dose biotin: Can interfere with immunoassays—stop 72 hours before testing
- Topical testosterone application: Don't apply to blood draw site for 24+ hours
Understanding Your Testosterone Results
Interpreting testosterone levels requires understanding both population-based reference ranges and individual clinical context. A single number alone doesn't determine whether you need treatment.
Standard Reference Ranges
| Measurement | Reference Range |
|---|---|
| Total Testosterone (AUA) | 300-1,000 ng/dL |
| Total Testosterone (Endocrine Society) | 264-916 ng/dL (CDC harmonized) |
| Free Testosterone | 5-21 pg/mL (varies by method) |
| Low Testosterone Threshold | <300 ng/dL (AUA) or <264 ng/dL (ES) |
Age-Specific Reference Ranges
A landmark 2022 study published in the Journal of Urology analyzed testosterone levels in 1,486 healthy men aged 20-44 and established age-specific thresholds for testosterone deficiency, challenging the one-size-fits-all 300 ng/dL cutoff:9
| Age Group | 2.5th Percentile | Clinical Implication |
|---|---|---|
| 20-24 years | 409 ng/dL | Young men need higher levels |
| 25-29 years | 413 ng/dL | Peak testosterone years |
| 30-34 years | 359 ng/dL | Early decline begins |
| 35-39 years | 352 ng/dL | 1-2% annual decline |
| 40-44 years | 350 ng/dL | Approaching standard threshold |
| 45+ years | ~300 ng/dL | Standard threshold appropriate |
Clinical significance: A 28-year-old man with a testosterone level of 350 ng/dL is likely experiencing hormonal insufficiency, even though he technically exceeds the standard 300 ng/dL threshold. This underscores the importance of clinical context and symptom correlation in diagnosis.
The Confirmation Requirement
Because testosterone levels can fluctuate significantly day-to-day, the Endocrine Society requires confirmation on a second morning sample before diagnosing hypogonadism.1 Approximately 30% of men with an initially low testosterone level will have normal concentrations when retested.
Both tests should be performed:
- In the morning (7-10 AM)
- In a fasting state
- At least one week apart
- When not acutely ill
- Ideally at the same laboratory for consistency
The Complete Hormone Panel: Beyond Testosterone
A testosterone level alone doesn't tell the complete story. Understanding why testosterone is low is crucial for determining the appropriate treatment approach. Additional hormone testing helps differentiate between primary and secondary hypogonadism and identifies underlying conditions that may require treatment.
Luteinizing Hormone (LH)
LH is produced by the pituitary gland and signals the Leydig cells in the testes to produce testosterone. LH levels help distinguish the location of the problem:10
- High LH + Low Testosterone = Primary Hypogonadism (testicular failure)
- Low/Normal LH + Low Testosterone = Secondary Hypogonadism (pituitary/hypothalamic problem)
- This distinction matters because secondary hypogonadism may be reversible and fertility can potentially be restored with gonadotropin therapy
Follicle-Stimulating Hormone (FSH)
FSH regulates sperm production (spermatogenesis). In primary hypogonadism, FSH is particularly important because it has a longer half-life and demonstrates less variability than LH, making it more sensitive for detecting testicular failure.10
- Elevated FSH suggests impaired spermatogenesis
- Very high FSH levels warrant karyotype testing to rule out Klinefelter syndrome
- FSH baseline is essential before starting any testosterone therapy if fertility preservation is a concern
Sex Hormone-Binding Globulin (SHBG)
SHBG is the carrier protein that tightly binds testosterone, controlling how much is available to tissues. SHBG levels affect the interpretation of total testosterone results:
- Low SHBG (common in obesity, diabetes) → Total testosterone may appear low despite adequate free testosterone
- High SHBG (common with aging, liver disease) → Total testosterone may appear normal despite low free testosterone
- Measuring SHBG allows accurate calculation of free and bioavailable testosterone
Prolactin
Prolactin is measured when secondary hypogonadism is suspected. Elevated prolactin (hyperprolactinemia) suppresses GnRH and LH, leading to low testosterone. Causes include:11
- Prolactinoma: Pituitary tumor that produces prolactin
- Medications: Antipsychotics, certain antidepressants, metoclopramide
- Hypothyroidism: Can elevate prolactin levels
- Prolactin >30 ng/mL typically warrants pituitary MRI imaging
Additional Tests to Consider
Depending on clinical presentation, additional testing may include:
- Estradiol (E2): Evaluates testosterone-to-estrogen conversion; elevated in obesity
- PSA: Baseline prostate screening before testosterone therapy
- Hemoglobin/Hematocrit: Testosterone increases red blood cell production; baseline and monitoring required
- Thyroid function (TSH, Free T4): Thyroid disorders affect testosterone metabolism
- Lipid panel: Testosterone affects lipid metabolism
- Iron/Ferritin/Transferrin: Screen for hemochromatosis in secondary hypogonadism
- Cortisol: Adrenal function if panhypopituitarism suspected
- Semen analysis: If fertility is a concern
At-Home Testing vs. Clinical Laboratory Testing
The rise of direct-to-consumer health testing has made at-home testosterone kits increasingly popular. Understanding the limitations of these tests is essential for making informed decisions about your health.
At-Home Testing Methods
Saliva Testing:
- Only measures free testosterone (not total or SHBG-bound)
- Easily contaminated by food, blood from gum inflammation, or oral products
- Studies show inconsistent correlation with blood levels
- Not recommended by any major endocrine society for clinical diagnosis
Finger-Prick Dried Blood Spot (DBS):
- Small sample volume can affect accuracy
- Hematocrit variations affect results
- One study showed home test results varied 26-41% in men compared to clinical standards12
- May provide reasonable screening but requires clinical confirmation
Clinical Laboratory Testing
Clinical testing through venous blood draw remains the standard of care for testosterone diagnosis:
- Larger sample volume improves accuracy
- Access to CDC-certified assays and LC-MS/MS methodology
- Complete hormone panel available (LH, FSH, SHBG, prolactin)
- Results interpreted by qualified medical providers
- Direct integration with treatment planning
Bottom line: At-home tests may provide general insight into your hormone status, but abnormal results should always be confirmed with clinical laboratory testing before any treatment decisions are made.
After Your Results: Understanding the Path Forward
Once you have confirmed low testosterone levels, the next step is determining whether treatment is appropriate and which approach best addresses your individual situation.
Diagnosis Requires Both Lab Values AND Symptoms
The Endocrine Society explicitly states that hypogonadism should only be diagnosed in men with both symptoms consistent with testosterone deficiency and unequivocally low testosterone levels.1 A low number alone doesn't automatically indicate need for treatment—some men with low levels are asymptomatic, while others with borderline levels experience significant symptoms.
Treatment Options Depend on Diagnosis Type
For Primary Hypogonadism (testicular failure):
- Testosterone replacement therapy (TRT) is the primary treatment option
- Options include injections, topical gels, patches, or pellets
- Fertility preservation may require sperm banking prior to treatment
For Secondary Hypogonadism (pituitary/hypothalamic):
- Address underlying cause if reversible (medication-induced, obesity, sleep apnea)
- Fertility-preserving options: clomiphene citrate, enclomiphene, hCG
- TRT if underlying cause cannot be corrected and fertility not a concern
- Pituitary imaging if prolactin elevated or testosterone severely low (<150 ng/dL)
Contraindications to Testosterone Therapy
The Endocrine Society recommends against starting testosterone therapy in patients with:1
- Active desire for fertility (unless using combination therapy)
- Known or suspected prostate cancer
- Breast cancer
- Hematocrit >50% (polycythemia)
- Untreated severe obstructive sleep apnea
- Uncontrolled heart failure
- PSA >4 ng/mL or palpable prostate abnormality without urological evaluation
Ready to Get Tested?
At Arsenal Men's Health, we provide comprehensive testosterone testing using CDC-standardized, CLIA-certified laboratory analysis with LC-MS/MS methodology for maximum accuracy. Our complete hormone panel includes total and free testosterone, LH, FSH, SHBG, prolactin, and additional markers as clinically indicated.
As a nurse practitioner-owned Utah telehealth clinic, we specialize in discreet, convenient hormone optimization for men across the state. Your consultation includes expert interpretation of results and personalized treatment recommendations based on evidence-based clinical guidelines.
Book Your Free Consult (385) 666-6292Peer-Reviewed References
- Bhasin S, Brito JP, Cunningham GR, et al. Testosterone Therapy in Men With Hypogonadism: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2018;103(5):1715-1744.
- Mulhall JP, Trost LW, Brannigan RE, et al. Evaluation and Management of Testosterone Deficiency: AUA Guideline. J Urol. 2018;200(2):423-432.
- Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999;84(10):3666-3672.
- Vesper HW, Botelho JC, Wang Y. Challenges and improvements in testosterone and estradiol testing. Asian J Androl. 2014;16(2):178-184.
- Wang C, Catlin DH, Demers LM, et al. Measurement of total serum testosterone in adult men: comparison of current laboratory methods versus liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab. 2004;89(2):534-543.
- Guidelines & Protocols Advisory Committee. Testosterone Testing Protocol. British Columbia Ministry of Health. 2023.
- Rubinstein AL, Carpenter DM. Association Between Commonly Prescribed Opioids and Androgen Deficiency in Men. Pain Med. 2014;15(10):1697-1705.
- Leproult R, Van Cauter E. Effect of 1 week of sleep restriction on testosterone levels in young healthy men. JAMA. 2011;305(21):2173-2174.
- Dupree JM, Langille GM, Gorbachinsky I, et al. Age-Specific Thresholds for the Diagnosis of Testosterone Deficiency in Men 20-44 Years of Age. J Urol. 2022;207(4):853-859.
- Petak SM, Nankin HR, Spark RF, et al. American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Evaluation and Treatment of Hypogonadism in Adult Male Patients. Endocr Pract. 2002;8(6):440-456.
- Melmed S, Casanueva FF, Hoffman AR, et al. Diagnosis and treatment of hyperprolactinemia: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96(2):273-288.
- Salamin O, Nicoli R, Xu C, et al. Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women. J Pharm Biomed Anal. 2021;204:114280.
- Lincoff AM, Bhasin S, Flevaris P, et al. Cardiovascular Safety of Testosterone-Replacement Therapy. N Engl J Med. 2023;389(2):107-117.
- Sizar O, Schwartz J. Hypogonadism. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024.
