Testosterone, Free (Equilibrium Dialysis) and Total (LC-MS/MS) Panel
This panel measures total testosterone by LC-MS/MS together with free testosterone by equilibrium dialysis, the reference-standard combination for androgen assessment. Directly measured free testosterone is the most reliable index of bioavailable androgen when SHBG is abnormal, as in obesity, insulin resistance, ageing, or oestrogen exposure, where total testosterone alone can mislead.
2
Biomarkers
Blood (serum)
Sample
3–6 business days
Turnaround
Preferably yes — an early-morning fasting sample is recommended in men because of diurnal variation and the effect of glucose intake on testosterone
Fasting
What is the Free + Total Testosterone (Dialysis/MS) test?
This panel measures total testosterone by mass spectrometry together with free testosterone by equilibrium dialysis — the reference-standard combination for androgen assessment. It provides both the overall androgen pool and the directly measured bioavailable fraction, resolving the ambiguity that arises when total testosterone alone is interpreted in the presence of abnormal binding proteins. Within androgen assessment, total testosterone is the first-line measure, but it reflects bound and free hormone together and can misrepresent bioavailable androgen whenever SHBG is altered — common in obesity, insulin resistance, ageing, thyroid or hepatic disease, and with oestrogen exposure. Adding a directly measured free testosterone resolves this. Equilibrium dialysis physically separates the free fraction and is the reference method: it is materially more reliable than analogue immunoassay free testosterone, which is not recommended, and avoids the assumptions of calculated free testosterone. This panel is therefore indicated where accurate assessment of bioavailable androgen matters — borderline or discordant total testosterone, suspected SHBG abnormality, and evaluation of hypogonadism or hyperandrogenism where the clinical picture and total do not align.
What does the Free + Total Testosterone (Dialysis/MS) test measure?
Testosterone is the principal androgen, produced mainly by testicular Leydig cells in men and by the ovaries and adrenal glands in women. Total testosterone comprises hormone bound tightly to sex hormone-binding globulin (SHBG), loosely bound to albumin, and a small free fraction. Secretion is pulsatile with a diurnal rhythm peaking in the early morning in men. Measured here by liquid chromatography-tandem mass spectrometry, the reference-standard method offering specificity across the wide concentration range.
Total testosterone is the primary measure of androgen status and the first-line result in this panel. However, because it reflects bound and free hormone together, it can misrepresent bioavailable androgen when SHBG is abnormal — which is precisely why it is paired here with a directly measured free testosterone.
Free testosterone is the small unbound fraction — roughly 1–2% of total — that is not attached to SHBG or albumin and is directly available to tissues. Equilibrium dialysis physically separates the free fraction across a semipermeable membrane before quantification, which is why it is regarded as the reference method. It is independent of binding-protein concentration, unlike total testosterone.
Directly measured free testosterone by equilibrium dialysis is the most reliable index of bioavailable androgen, particularly when SHBG is abnormal — as in obesity, insulin resistance, ageing, thyroid disease, hepatic disease, or oestrogen exposure — where total testosterone can be misleading. It is materially more accurate than analogue immunoassay free testosterone, which is unreliable and should not be used, and it avoids the assumptions inherent in calculated free testosterone.
Why is the Free + Total Testosterone (Dialysis/MS) test ordered?
- Investigation of male hypogonadism where the total testosterone is borderline or discordant with the clinical picture, and bioavailable androgen must be established
- Assessment of androgen status where SHBG is likely abnormal — obesity, insulin resistance, ageing, thyroid disease, hepatic disease, or oestrogen exposure
- Evaluation of hyperandrogenism in women, where free testosterone is often the more sensitive marker and mass-spectrometry accuracy at low levels is essential
- Workup of polycystic ovary syndrome where free testosterone adds sensitivity over total testosterone alone
- Monitoring of testosterone replacement therapy where bioavailable androgen is being titrated
- Situations where a previously reported analogue-immunoassay or calculated free testosterone is unreliable and a reference-method measurement is required
- Assessment of unexplained androgenic or hypogonadal symptoms where total testosterone alone has not clarified the picture
Sample collection and turnaround
Sample type
Blood (serum)
Fasting required
Preferably yes — an early-morning fasting sample is recommended in men because of diurnal variation and the effect of glucose intake on testosterone
Collection method
Venipuncture at patient service centre
Turnaround
3–6 business days
Collection notes
Collect in the early morning, ideally fasting, particularly in men, and confirm a low male result on a repeat early-morning sample. Document testosterone or anabolic therapy, opioids, glucocorticoids, and any acute illness. Note that equilibrium-dialysis free testosterone is method-specific — use the same method for serial monitoring.
Specimen requirements
Serum, with sufficient volume for both the mass-spectrometry total and the equilibrium-dialysis free testosterone per Quest protocol. Separate serum from cells; specimen stable refrigerated. Follow Quest transport guidelines.
What can affect Free + Total Testosterone (Dialysis/MS) results?
Testosterone peaks in the early morning in men and declines through the day, so a low afternoon value may not be representative. An early-morning, ideally fasting, sample is recommended, and a low male result confirmed on repeat.
Conditions raising SHBG (oestrogens, hyperthyroidism, ageing, hepatic disease) or lowering it (obesity, insulin resistance, hypothyroidism) shift total testosterone without necessarily changing free testosterone — the central reason this panel measures the free fraction directly.
Free testosterone by equilibrium dialysis is not interchangeable with calculated or analogue-immunoassay free testosterone; results and reference intervals differ by method, so serial monitoring must use the same method.
Acute illness, significant stress, and recent vigorous exercise can transiently lower testosterone, so measurement is best deferred to a stable, representative day.
Opioids, glucocorticoids, and some other drugs suppress the hypothalamic-pituitary-gonadal axis or androgen production, lowering testosterone independent of primary gonadal disease. Medication history should be documented.
Administered testosterone and anabolic agents raise measured testosterone and suppress endogenous production; therapy and timing relative to dosing must be documented for meaningful interpretation.
What do Free + Total Testosterone (Dialysis/MS) results mean?
Elevated
In women, an elevated free testosterone supports hyperandrogenism and is often more sensitive than the total; markedly elevated values raise concern for an androgen-secreting tumour. In men, elevation may reflect exogenous androgen use. Total and free are interpreted together — a raised total with a normal free may simply reflect a high SHBG.
Normal
Normal total and free testosterone on a correctly timed sample suggest normal androgen status. Where the total is borderline but the free is clearly normal, bioavailable androgen is adequate and an SHBG effect on the total is the likely explanation; the converse pattern warrants attention.
Reduced
In men, a low free testosterone by dialysis, on an early-morning sample and confirmed on repeat, supports hypogonadism even when the total is borderline — a common and clinically important pattern in obesity and insulin resistance, where a low SHBG lowers total testosterone while free may be preserved, or vice versa. LH and FSH then localise the cause as primary or central.
Note: Free testosterone by equilibrium dialysis is the reference method and is not interchangeable with calculated or analogue-immunoassay free testosterone — the latter is unreliable and should not be used. Results must be interpreted by sex, age, and collection time, and total and free read together. Diurnal rhythm, illness, fasting status, and medications all affect results, and a single low male value should be confirmed on an early-morning repeat.
Frequently asked questions
Total testosterone reflects bound and free hormone together, so it can misrepresent bioavailable androgen when SHBG is abnormal. Directly measured free testosterone reflects the fraction actually available to tissues and resolves this ambiguity.
How to access the Free + Total Testosterone (Dialysis/MS) test
The Free + Total Testosterone (Dialysis/MS) reference page is free and open to all practitioners. Create a free practitioner account to see pricing, save tests to your list, and generate requisition forms. Verification takes 1–2 business days.
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Published: June 2, 2026
Last reviewed: June 2, 2026
- Bhasin S, et al. Testosterone Therapy in Men With Hypogonadism: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. (2018)
- Rosner W, et al. Position statement: Utility, limitations, and pitfalls in measuring testosterone. J Clin Endocrinol Metab. (2007)
- Goldman AL, et al. A Reappraisal of Testosterone's Binding in Circulation. Endocr Rev. (2017)
- Travison TG, et al. Harmonized Reference Ranges for Circulating Testosterone Levels in Men. J Clin Endocrinol Metab. (2017)
This content is for licensed healthcare practitioners only and does not constitute medical advice. Clinical decisions should be based on the full clinical picture and not on any single test result.
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