What Is Equilibrium Dialysis Free Testosterone Testing?

Equilibrium dialysis free testosterone testing is a laboratory method that physically separates unbound testosterone from protein-bound testosterone in a blood sample, then quantifies the free fraction using mass spectrometry. The Endocrine Society's 2018 clinical practice guideline identifies equilibrium dialysis (or a calculation formula validated against it) as the recommended approach for measuring free testosterone when total testosterone falls near the lower limit of normal or when sex hormone-binding globulin (SHBG) is altered (Bhasin et al., 2018). For men evaluating hormone health through lab testing, understanding why the method behind the number matters is part of getting an accurate clinical picture.

Equilibrium dialysis free testosterone testing uses a semipermeable membrane to separate unbound testosterone from proteins like SHBG and albumin in serum. The Endocrine Society recommends this method, or a formula validated against it, when total testosterone is borderline or SHBG levels are abnormal. It is the reference measurement against which other free testosterone assays are compared.

Less than 3% of circulating testosterone is unbound. The rest binds to SHBG (roughly 50-60%) and albumin (roughly 40-50%). That unbound fraction is generally considered the portion most readily available to interact with androgen receptors. A 2018 study by Fiers and colleagues, comparing direct equilibrium dialysis with LC-MS/MS against several calculation formulas, confirmed that free testosterone represents only a small fraction of total testosterone in the male cohorts studied (Fiers et al., 2018). When SHBG shifts up or down, the relationship between total testosterone and free testosterone changes, and the measurement method used to detect that shift becomes clinically relevant.

How Does Equilibrium Dialysis Measure Free Testosterone?

The laboratory process works by placing a serum sample on one side of a semipermeable membrane and a protein-free buffer on the other. Over several hours at body temperature (37 degrees Celsius), unbound testosterone molecules pass through the membrane until concentrations on both sides reach equilibrium. The buffer side then contains only free testosterone, which the lab quantifies using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The CDC's Clinical Standardization Programs have been developing a high-throughput version of this method, called ED-ID-UHPLC-MS/MS, using a custom dialyzer with a 1:1 sample-to-buffer volume and HEPES buffer at pH 7.4 (CDC CSP). Reported analytical performance from this work includes a limit of detection of 0.6 pg/mL, accuracy within 5%, and precision below 10% coefficient of variation.

That level of measurement precision matters. Free testosterone concentrations are very low relative to total testosterone, often in the single-digit pg/mL range for some populations. A method that can't reliably measure at those concentrations may misclassify borderline cases.

Why the Testing Method Matters

Not all free testosterone results are interchangeable. A 2024 CDC interlaboratory comparison found that immunoassay-based free testosterone results averaged roughly 6 times lower than equilibrium dialysis with LC-MS/MS results from the same samples. Agreement within each method group was relatively close, but the gap between groups was large. Calculated free testosterone using the Vermeulen equation overestimated the equilibrium dialysis result by an average of 15%.

Fiers and colleagues documented the same pattern in a study of 183 women and 146 men. Median ratios of calculated free testosterone to directly measured ED free testosterone were 1.19 for the Vermeulen formula, 1.00 for the Ly formula, and 2.05 for the Zakharov formula (Fiers et al., 2018). The Vermeulen formula showed the least dependence on SHBG, albumin, and total testosterone among the tested equations, but it still overestimated.

These aren't small academic differences. A man with borderline total testosterone whose calculated free testosterone reads "normal" on one formula might have a result that reads "low" when measured by equilibrium dialysis. Two providers looking at two different methods could reach two different clinical conclusions from the same blood sample. The Endocrine Society explicitly warns against using direct analog-based free testosterone immunoassays, calling them inaccurate (Bhasin et al., 2018).

No harmonized reference range for free testosterone exists across laboratories. Each lab's range depends on the method it uses, and switching methods or labs between serial measurements can create the appearance of a change that's actually an artifact.

Equilibrium Dialysis vs. Calculated and Immunoassay Methods

The table below compares the 3 common approaches to measuring free testosterone.

For men with normal SHBG and a total testosterone result well above or well below the guideline-defined lower limit, the measurement method is less likely to change the clinical picture. The method matters most at the borderline, where a small difference in the number could shift interpretation. A man with borderline total testosterone and elevated SHBG is exactly the case where a calculated result might mask low free testosterone.

When Should a Provider Order This Test?

The Endocrine Society's 2018 guideline specifies 2 situations where free testosterone measurement adds clinical value beyond total testosterone alone. The first is when total testosterone falls near the lower limit of normal. The second is when the individual has a condition that alters SHBG concentrations (Bhasin et al., 2018).

The AUA's 2018 guideline uses a defined total testosterone threshold on 2 separate early-morning fasting measurements as a reasonable diagnostic cutoff when symptoms are present. Free testosterone is not the primary diagnostic measure under the AUA framework, but it can clarify borderline situations (Mulhall et al., 2018). A provider determines which thresholds apply based on individual presentation.

In clinical practice, the free testosterone by dialysis test is most useful for men whose total testosterone falls in a borderline range, especially if SHBG is elevated or suppressed. A high SHBG binds more testosterone and reduces the free fraction. A low SHBG does the opposite, sometimes making total testosterone look normal while symptoms persist.

Monitoring during testosterone replacement therapy is another situation where a provider may order this test, with timing and panel composition individualized to the treatment plan. A man on treatment whose total testosterone falls within the target range but continues to report symptoms may have a mismatch between total and free levels, sometimes driven by SHBG changes over the course of treatment.

Conditions That Alter SHBG and Shift Free Testosterone

SHBG production rises or falls in response to several conditions, and each shift tends to move the free testosterone fraction in a generally expected direction.

Conditions associated with decreased SHBG (which increase the free fraction relative to total testosterone): obesity, type 2 diabetes, glucocorticoid use, certain progestins, androgenic steroids, nephrotic syndrome, hypothyroidism, acromegaly, and certain SHBG gene polymorphisms. The Endocrine Society guideline lists these specifically as conditions that alter SHBG binding capacity (Bhasin et al., 2018).

Conditions associated with increased SHBG (which decrease the free fraction): aging, liver disease, hyperthyroidism, estrogen therapy, and anticonvulsant use. In these cases, total testosterone can appear normal while the biologically available fraction is low.

When SHBG levels are abnormal, a calculated free testosterone that relies on assumptions about binding kinetics may diverge from the actual unbound concentration. This is the exact scenario where equilibrium dialysis adds the most clinical clarity.

A common example: an obese man with low SHBG and a total testosterone near the guideline-defined lower limit. His calculated free testosterone might look normal because the low SHBG leaves more testosterone unbound. But his total testosterone may still fall below the threshold a provider uses, and relying on the calculated free T alone might not give the full picture.

What Do Equilibrium Dialysis Free Testosterone Results Mean?

Reference ranges for percent free testosterone by equilibrium dialysis vary by laboratory and assay platform. No universal reference range exists, a consistent finding across the Endocrine Society guideline and the CDC's interlaboratory work. A provider interprets the result using the reporting lab's own range alongside total testosterone and SHBG.

A result below the lab's reference range, paired with a low or borderline total testosterone on repeat fasting morning draws, may warrant further evaluation. Symptoms that commonly prompt that evaluation include reduced libido, fatigue, reduced energy, loss of lean mass, and depressed mood. A provider considers these alongside consistently low lab values, not a single number in isolation, as part of a shared discussion about symptoms, goals, and risks.

Interpreting any free testosterone result without the matching total testosterone and SHBG is incomplete. A low percent free testosterone with a normal total testosterone and high SHBG tells a different story than a low percent free testosterone with a low total testosterone and normal SHBG. The first may point toward an SHBG-driven mechanism. The second may suggest reduced production, though further evaluation would clarify the cause.

Results should be discussed with a licensed provider who can interpret the values in context. Ordering a testosterone testing panel that includes total testosterone, free testosterone by equilibrium dialysis, and SHBG gives a more complete initial picture.

Lab Tests That Pair With Equilibrium Dialysis Free Testosterone

Free testosterone by dialysis doesn't exist in a vacuum. The biomarkers listed below are commonly ordered alongside it, and each one adds a different layer of context to the hormone picture.

Total testosterone (LC-MS/MS): The first-line test. Total T is still the starting point per both the AUA and Endocrine Society guidelines. Free T is the second step when total T is borderline or SHBG is suspect.

SHBG: Without SHBG, the free testosterone result lacks a reference frame. SHBG is one factor that may explain why free T is high or low relative to total T.

Estradiol (sensitive, LC-MS/MS): Testosterone aromatizes to estradiol. Tracking sensitive estradiol alongside testosterone provides a measure of that conversion.

LH and FSH: These pituitary hormones distinguish primary hypogonadism (testicular) from secondary hypogonadism (pituitary or hypothalamic). A low total testosterone with low LH and FSH suggests a secondary cause, which changes the evaluation and management path.

Hematocrit (CBC): The AUA guideline lists hematocrit among biomarkers tracked during therapy. A complete blood count that includes hematocrit is standard before starting and during any testosterone protocol.

Prolactin: Elevated prolactin can suppress LH and secondarily lower testosterone. Testing prolactin may help identify a contributing factor when LH is low.

Ordering individual markers one at a time is one approach. Pre-built lab bundles group the relevant markers together, which reduces the number of separate draws and covers the markers that clinical guidelines recommend checking as a set. Men interested in where to start can review how testing works and select the panel that fits their situation.

The question clients should ask before ordering: which free testosterone method (equilibrium dialysis, ultrafiltration, calculated formula, or analog immunoassay) will the lab use, and what is the lab-specific reference range? Knowing the method matters as much as knowing the number. Equilibrium dialysis free testosterone, paired with total testosterone and SHBG through a Pre-TRT Panel or similar grouping, gives a provider a strong starting point for evaluating hormone status.

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

Is equilibrium dialysis more accurate than calculated free testosterone?

The Endocrine Society's 2018 guideline recommends equilibrium dialysis or a calculation validated against it when free testosterone is clinically indicated. Fiers et al. (2018) found that the Vermeulen formula overestimates equilibrium dialysis results by a median factor of 1.19, while the Zakharov formula overestimates by a median factor of 2.05. The accuracy gap widens when SHBG is abnormal.

Why do free testosterone results differ between labs?

No harmonized free testosterone reference range exists across laboratories. CDC interlaboratory comparisons have found that immunoassay-based methods average roughly 6 times lower than equilibrium dialysis with LC-MS/MS. Switching labs or methods between serial tests can create the appearance of a change that is actually a measurement artifact.

When does the Endocrine Society recommend measuring free testosterone?

The Endocrine Society's 2018 guideline recommends free testosterone measurement in 2 situations: when total testosterone falls near the lower limit of normal, and when a condition alters SHBG (obesity, diabetes, thyroid disease, liver disease, certain medications). Analog immunoassays should not be used for this purpose.

Can a normal calculated free testosterone result still miss a deficiency?

Yes. Because calculated values can overestimate relative to equilibrium dialysis, a man with borderline total testosterone and altered SHBG may show a "normal" calculated free testosterone that would fall below the reference range if measured by dialysis. Guidelines treat free testosterone as an adjunct in specific settings, not a standalone replacement for total testosterone plus symptoms.

What does percent free testosterone mean on my lab report?

Percent free testosterone is the proportion of total testosterone that is unbound to proteins in the blood. The reference range varies by laboratory and assay platform, so there is no single universal number to compare against. It should be interpreted alongside total testosterone and SHBG using the reporting lab's own range.

Should I ask my provider which free testosterone method the lab uses?

Yes. The Endocrine Society explicitly advises against direct analog immunoassays and recommends equilibrium dialysis or an accurate formula validated against it. Asking which method the lab uses, along with its specific reference range, helps avoid misinterpretation of borderline results.

Does Marek Diagnostics offer equilibrium dialysis free testosterone testing?

Marek Diagnostics offers a free testosterone by equilibrium dialysis test paired with total testosterone by LC-MS/MS. This combination follows the measurement approach recommended by the Endocrine Society's 2018 guideline and can be ordered individually or as part of a pre-built lab panel.

References

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

• J Clin Endocrinol Metab. 2018;103(6):2167-2174

• J Urol. 2018;200(2):423-432

• CDC Clinical Standardization Programs, Hormones