Biomarkers To Monitor While On Testosterone Therapy

Testosterone replacement therapy (TRT) requires scheduled blood work and safety checks at every stage of treatment. The Endocrine Society and the American Urological Association (AUA) both publish monitoring protocols that cover the same core labs: total testosterone, hematocrit, prostate-specific antigen (PSA) in eligible men, blood pressure, and lipids (Bhasin et al., 2018; Mulhall et al., 2018). Skipping those labs leaves rising red blood cell counts, prostate marker changes, and cardiovascular shifts undetected until symptoms appear. Monitoring is not optional for men on a Schedule III controlled substance.

Direct-pay lab testing gives clients a way to track these markers on the schedule their provider sets, without insurance gatekeeping the panel. The goal is simple in principle but layered in practice: confirm the medication is working, confirm it isn't causing harm, and catch changes early enough to adjust the protocol before a problem becomes a complication.

Biomarkers monitored during testosterone therapy fall into two groups. The first group, covered by both U.S. guidelines, includes safety labs that carry clear intervention thresholds (hematocrit above 54%, PSA rises above defined limits). The second group includes metabolic and hormonal markers (estradiol, lipids, metabolic panel) that provide context for dose decisions and long-term health but don't carry the same guideline-mandated stop rules. Understanding which category each lab belongs to changes how results are interpreted.

Why Does TRT Require Regular Lab Monitoring?

Testosterone affects more than circulating hormone levels. It stimulates erythropoiesis (red blood cell production), alters lipid metabolism, may raise blood pressure, and can suppress the hypothalamic-pituitary-gonadal axis, reducing LH, FSH, and sperm production (Bhasin et al., 2018). Each of those downstream effects carries a monitoring requirement.

The TRAVERSE trial, the largest randomized safety study of testosterone therapy to date (5,246 men, mean follow-up approximately 33 months), met its prespecified noninferiority endpoint for major adverse cardiac events (MACE) compared to placebo (7.0% vs 7.3%; HR 0.96; 95% CI 0.78-1.17) (Lincoff et al., 2023). That result led the FDA to remove the prior cardiovascular boxed-warning language from testosterone product labels in February 2025 (FDA, 2025).

The neutral cardiac headline, though, came with secondary findings that matter for monitoring. TRAVERSE also reported higher rates of atrial fibrillation (3.5% vs 2.4%, P=0.02), acute kidney injury (2.3% vs 1.5%, P=0.04), and pulmonary embolism (0.9% vs 0.5%) in the testosterone group (Lincoff et al., 2023). None of those secondary signals cancel the primary result, but they reinforce why hematocrit, blood pressure, and kidney function belong on every monitoring visit.

Which Biomarkers Should Be Tested During TRT?

Both the Endocrine Society and the AUA agree on the core monitoring panel, though they differ on specific thresholds and timing. The following biomarkers represent the guideline-minimum labs plus the markers added by the 2025 FDA label update.

Total and Free Testosterone

Total testosterone confirms absorption and documents whether the dose places the client in the intended range. The AUA describes a conditional target in the middle tertile of the lab's reference interval, around 450-600 ng/dL for most assays (Conditional Recommendation, Evidence Level Grade C) (Mulhall et al., 2018). The Endocrine Society uses a "mid-normal" framing and, for men on cypionate or enanthate, has referenced a 400-700 ng/dL window measured about one week post-injection (Bhasin et al., 2018).

These are general reference points from each society, not fixed targets for every individual. A provider evaluates the full picture, including symptoms, free testosterone, and SHBG.

Free testosterone measurement becomes necessary when total testosterone sits near the diagnostic cutoff or when a condition alters sex hormone-binding globulin (SHBG). Obesity and type 2 diabetes lower SHBG, making total testosterone look low while free testosterone may not be. Aging and hyperthyroidism raise SHBG, creating the opposite pattern. The Endocrine Society recommends free testosterone by equilibrium dialysis or an accurate calculation formula, not by direct analog immunoassay, when SHBG is abnormal (Bhasin et al., 2018).

Assay consistency also matters. The CDC Hormone Standardization (HoSt) program exists because nonstandardized assays can classify the same blood sample differently (CDC, 2024). The Endocrine Society's diagnostic cutoff for "unequivocally low" total testosterone on a CDC-standardized assay is 264 ng/dL. The AUA uses 300 ng/dL. A man can meet one society's definition and fall outside the other's without any change in his biology.

Hematocrit and Hemoglobin

Hematocrit is the most consistent safety recommendation across all guidelines and the lab most likely to trigger a treatment hold.

Testosterone stimulates red blood cell production. In most men, erythropoiesis rises during the first 1-6 months of treatment and then plateaus. The AUA advises not starting testosterone if baseline hematocrit exceeds 50% until the cause is evaluated. On treatment, hematocrit at or above 54% requires intervention in both the Endocrine Society and AUA frameworks: dose reduction, temporary hold, evaluation for sleep apnea or hypoxia, or therapeutic phlebotomy (Bhasin et al., 2018; Mulhall et al., 2018).

Formulation choice affects hematocrit risk. A review by Ohlander and colleagues found that short-acting intramuscular cypionate or enanthate produced the highest rates of hematocrit exceeding 50% (66.7% across reviewed studies), compared to 35.1% for pellets, 12.8% for transdermal gel, and 7% for long-acting intramuscular undecanoate (Ohlander et al., 2018). Marek Diagnostics does not offer pellets; they are included here only for published comparison data. A later network meta-analysis confirmed all formulations raise mean hematocrit versus placebo, with intramuscular cypionate and enanthate producing larger increases than patch (Nackeeran et al., 2022). TRAVERSE's low rate of hematocrit-related discontinuation occurred on titrated gel with a central monitoring algorithm, not on self-injected cypionate at fixed doses.

Prostate-Specific Antigen (PSA)

Both guidelines require a baseline PSA in men aged 40 and older before starting testosterone. The Endocrine Society calls for urologic referral if PSA rises more than 1.4 ng/mL in any 12-month period, if PSA velocity exceeds 0.4 ng/mL per year after the 6-month on-treatment value, if confirmed PSA exceeds 4.0 ng/mL, or if a prostate abnormality is found on exam (Bhasin et al., 2018).

TRAVERSE's prostate safety analysis found no statistically significant difference in high-grade prostate cancer between testosterone and placebo groups (5/2,596 vs 3/2,602; HR 1.62; 95% CI 0.39-6.77; P=0.51), though the confidence interval was wide and the trial was not designed to detect cancer differences (Bhasin et al., 2023). In the parent trial data, mean PSA rose more on testosterone than placebo (0.20 vs 0.08 ng/mL; P<0.001) (Lincoff et al., 2023). That finding is not a license to skip PSA monitoring. It is evidence that, in screened men without baseline prostate cancer, gel therapy over roughly 22 months did not raise high-grade cancer incidence to a statistically significant degree. Follow-up was not lifelong, and FDA labels still carry prostate-related warnings.

Estradiol (E2)

Estradiol testing is less routine than many articles suggest. The AUA recommends estradiol measurement if the client has baseline gynecomastia or breast symptoms, optionally at baseline in all men, and if breast symptoms develop on treatment. Estradiol should be measured in anyone taking an aromatase inhibitor (Mulhall et al., 2018). The Endocrine Society does not make routine estradiol monitoring a Grade 1 requirement (Bhasin et al., 2018).

There is no society-endorsed "ideal" male estradiol number on treatment. Providers who use a sensitive estradiol assay (LC-MS/MS) get more accurate readings at male-range concentrations than standard immunoassay provides. Blanket aromatase-inhibitor use to push estradiol to a specific number is not an Endocrine Society or AUA recommendation, and over-suppression of estradiol may worsen bone density, lipid profiles, and sexual function.

Blood Pressure

Blood pressure became a class-wide labeling concern with the February 2025 FDA update. After reviewing postmarket ambulatory blood pressure monitoring (ABPM) studies, the FDA required product-specific blood pressure information on testosterone labels (FDA, 2025). The 2018 guidelines from both societies predate this label change and do not list blood pressure as a formal TRT "lab," but the 2025 FDA action supports including blood pressure measurement at monitoring visits.

The magnitude of the blood pressure effect is product-specific. Providers should reference the individual product label rather than applying a single number across all formulations.

Lipid Panel

Testosterone therapy can shift lipid markers. An ancillary analysis from the Testosterone Trials (TTrials) found that 1 year of testosterone in older men produced small additional reductions compared to placebo in total cholesterol (approximately 6.1 mg/dL) and HDL (approximately 2.0 mg/dL), with a borderline LDL change (Mohler et al., 2018). The clinical significance of those small shifts is uncertain. The Endocrine Society lipid guideline advises against using testosterone as a treatment for dyslipidemia (Newman et al., 2020). ACC/AHA cholesterol guidelines govern statin decisions; testosterone does not replace that framework.

A lipid panel at baseline and then annually (or per the client's cardiovascular risk profile) is reasonable. Large drops in HDL or increases in LDL warrant a conversation with the prescribing provider about cardiovascular risk.

Metabolic Panel

A basic or comprehensive metabolic panel (CMP) measures liver enzymes (ALT, AST), kidney function (creatinine, BUN), blood glucose, and electrolytes. Current FDA-approved oral testosterone products (testosterone undecanoate) absorb through intestinal lymphatics and carry a different risk profile than the old 17-alpha-alkylated oral androgens, which were hepatotoxic. Injectable and transdermal routes have minimal direct liver toxicity signal.

TRAVERSE, however, found more acute kidney injury on testosterone than placebo (2.3% vs 1.5%, P=0.04) (Lincoff et al., 2023). That finding makes a metabolic panel at baseline and during follow-up reasonable, especially for men with diabetes, hypertension, or diuretic use. The CMP is not an AUA Table 7 required item, but it may be appropriate as part of a broader monitoring plan.

How Do the Two Major Guidelines Compare on TRT Monitoring?

The Endocrine Society and AUA agree on the core labs but differ on thresholds and cadence. The table below shows where they align and where they diverge.

Both societies agree the same man can test as "low" in one framework and "borderline" in another. The discrepancy does not mean one guideline is wrong. It means reference intervals and diagnostic cutoffs are not interchangeable, and providers should use the same certified assay at the same lab for every draw.

When Should Each Biomarker Be Tested?

Timing depends on the phase of treatment. The first 3-6 months carry the steepest risk for hematocrit changes, and the first 12 months carry the most PSA scrutiny. Once values stabilize, testing intervals widen.

Before starting TRT: Two morning fasting total testosterone samples (same lab, same assay), free testosterone and SHBG if total T is near the cutoff, CBC with hematocrit, PSA (men aged 40+), lipid panel, and metabolic panel. Blood pressure at the visit.

3-6 months after starting: Total testosterone, hematocrit, and PSA (if baseline was drawn). Blood pressure. The draw timing for testosterone depends on formulation. For injectable cypionate or enanthate, a mid-interval draw (roughly 3-4 days after injection) gives a representative level. A trough draw (just before the next injection) shows the lowest level. The provider may request one or both. For daily gels, draw at a consistent time relative to application.

12 months: Total testosterone, hematocrit, PSA, lipid panel, metabolic panel. Blood pressure.

Annually once stable: Total testosterone, hematocrit, PSA per age-appropriate guidelines, lipid panel. Blood pressure. The AUA recommends every 6-12 months when values are stable (Mulhall et al., 2018). More frequent testing may be appropriate during dose changes or if prior hematocrit values were elevated.

Consistency matters more than a single number. Drawing at the same time of day, at the same point in the dosing cycle, using the same lab, produces results that can be compared across visits. A single out-of-range value, drawn at trough instead of mid-cycle (or vice versa), doesn't always mean the dose is wrong.

When Do Lab Results Require Provider Attention?

Some lab results call for an immediate protocol change. Others call for closer follow-up.

Hematocrit at or above 54% is the clearest action trigger across both guidelines. Treatment should be held until hematocrit drops, and the provider should evaluate for sleep apnea, chronic hypoxia, or dehydration before restarting at a lower dose or switching formulations. A hematocrit of 50-53% is a watch zone, not a stop, but it warrants more frequent rechecks and possible dose or interval adjustments.

A confirmed PSA increase of more than 1.4 ng/mL in 12 months, or a confirmed PSA above 4.0
ng/mL, warrants urologic referral (Bhasin et al., 2018).

Testosterone levels well above 1,000 ng/dL without corresponding symptom improvement may indicate the dose exceeds what the client needs. More is not always better, and supraphysiologic levels may carry higher erythrocytosis and estrogen-conversion risk.

Estradiol-related symptoms (breast tenderness, fluid retention, mood changes) call for estradiol testing if it hasn't been done. Lab numbers alone, without symptoms, don't automatically require treatment. Over-suppressing estradiol with an aromatase inhibitor may do more harm than a moderately elevated level.

Liver enzyme elevation matters most for clients on oral testosterone formulations. For men on injectable or transdermal routes, liver enzymes are rarely affected by the testosterone itself, though they remain part of routine metabolic screening.

Which Additional Tests Apply in Specific Situations?

Some men need labs beyond the core panel.

LH and FSH are diagnostic labs, not routine monitoring labs. On exogenous testosterone, both will be suppressed because external testosterone signals the pituitary to stop producing gonadotropins. That suppression is expected. It is also the mechanism by which TRT reduces sperm production. The AUA recommends that men who may want children undergo a reproductive evaluation before starting therapy, and the guideline advises against prescribing exogenous testosterone to men actively trying to conceive (Strong Recommendation, Grade A) (Mulhall et al., 2018). Recovery of spermatogenesis after discontinuation is variable in both time and completeness.

Prolactin is indicated when testosterone is low and LH is low or low-normal, as this pattern may suggest a pituitary cause. It is not a routine TRT follow-up marker unless treating confirmed hyperprolactinemia.

SHBG helps interpret discrepancies between total testosterone and symptoms. A client with a total testosterone in range but persistent symptoms may have high SHBG binding most of the circulating hormone, leaving less free testosterone available.

Thyroid function may be relevant if fatigue or other symptoms persist despite testosterone levels in the intended range. Thyroid disease also alters SHBG and estradiol balance, which can affect TRT interpretation.

Bone mineral density (DXA) is recommended by the Endocrine Society after 1-2 years of treatment in men with osteoporosis or a history of low-trauma fracture. It is not a routine yearly blood test.

Biomarkers to monitor while on testosterone therapy don't end when symptoms improve. Hematocrit, blood pressure, and age-appropriate PSA remain safety labs after goals are met.

The AUA recommends lab checks every 6-12 months when stable. If symptoms resolve and testosterone is in range, but a client considers stopping, that discussion should include the exit plan: whether and how to adjust dosing, what to watch for, and how to reassess after discontinuation.

Disclaimer: This blog post is intended for informational purposes only and should not be considered medical advice. Always consult a healthcare professional before making changes to your health routine.

FAQs

What blood tests are required before starting testosterone therapy? 

Before starting TRT, the Endocrine Society and AUA both require two morning fasting total testosterone samples drawn at the same lab using the same assay. A complete blood count with hematocrit, a metabolic panel, and a lipid panel are standard baseline labs. Men aged 40 and older also need a baseline PSA. Free testosterone and SHBG testing is indicated when total testosterone sits near the diagnostic cutoff or when conditions like obesity or thyroid disease may alter SHBG levels (Bhasin et al., 2018).

How often should hematocrit be checked while on TRT? 

Both U.S. guidelines call for hematocrit at baseline, at 3-6 months after starting therapy, and then at least annually once values are stable. More frequent checks may be needed during the first year if the client is on intramuscular cypionate or enanthate, which produce higher erythrocytosis rates than gels or patches (Ohlander et al., 2018). Hematocrit at or above 54% requires treatment to be held and the cause evaluated.

Does testosterone therapy always raise PSA? 

Not always, but mean PSA rises more in men on testosterone than in matched controls. In the TRAVERSE parent trial data, mean PSA increased by 0.20 ng/mL in the testosterone group versus 0.08 ng/mL with placebo over roughly 22 months (Lincoff et al., 2023). High-grade prostate cancer incidence did not differ to a statistically significant degree between groups (Bhasin et al., 2023). A confirmed PSA rise of more than 1.4 ng/mL in 12 months, or a confirmed level above 4.0 ng/mL, warrants urologic referral per Endocrine Society guidelines.

Is estradiol testing required on every TRT blood panel? 

No. The AUA recommends estradiol testing when breast symptoms or gynecomastia are present, optionally at baseline, and for any client taking an aromatase inhibitor. The Endocrine Society does not list routine estradiol as a Grade 1 monitoring requirement. A sensitive LC-MS/MS estradiol assay is more accurate at male-range concentrations than standard immunoassay.

Did the FDA say testosterone is safe for the heart? 

The FDA removed boxed-warning language about excess cardiovascular events after TRAVERSE met its prespecified noninferiority endpoint for major adverse cardiac events (MACE) compared to placebo. At the same time, the February 2025 label update added new blood pressure warnings based on ambulatory monitoring data. TRAVERSE also showed higher rates of atrial fibrillation, acute kidney injury, and pulmonary embolism in the testosterone group (Lincoff et al., 2023; FDA, 2025). Monitoring did not become optional.

Can testosterone therapy affect fertility? 

Exogenous testosterone suppresses LH and FSH, which reduces or stops sperm production. The AUA advises against prescribing exogenous testosterone to men currently trying to conceive (Strong Recommendation, Grade A). Recovery of spermatogenesis after stopping is variable and not guaranteed. Men who may want children in the future should discuss fertility preservation strategies with their provider before starting treatment (Mulhall et al., 2018).

Why might total testosterone results differ between labs? 

Reference intervals and assay methods vary across laboratories. A total testosterone of 285 ng/dL could fall below the AUA's 300 ng/dL diagnostic cutoff but above the Endocrine Society's 264 ng/dL cutoff. The CDC's Hormone Standardization program certifies labs to reduce this variability (CDC, 2024). Using the same certified lab and the same assay for every draw produces the most reliable trend data.

References

• J Clin Endocrinol Metab. 2018;103(5):1715-1744

• J Urol. 2018;200(2):423-432

• N Engl J Med. 2023;389(2):107-117

• JAMA Netw Open. 2023;6(12):e2348692

• J Clin Endocrinol Metab. 2018;103(2):681-688

• J Clin Endocrinol Metab. 2020;105(12):dgaa674

• Sex Med Rev. 2018;6(1):77-85

• J Urol. 2022; Network Meta-Analysis

• FDA Class-Wide Labeling Changes. February 28, 2025

• CDC Hormone Standardization (HoSt) Program