Testosterone, Free, Mass Spectrometry/Equilibrium Dialysis

Free testosterone is the fraction of total testosterone that is not bound to circulating proteins. The free fraction is available to diffuse into target cells to interact with receptors or enzymes. The concentration of free testosterone is low relative to total testosterone, typically <3% of the total testosterone concentration. Total circulating testosterone is bound to sex hormone-binding globulin (SHBG) or albumin. Measurement of total and free testosterone is key in diagnosis of androgen excess or deficiency. Free testosterone is best measured by equilibrium dialysis, while total testosterone is measured by HPLC with tandem mass spectrometry. Free testosterone is estimated in this profile using these recommended techniques. Tritiated testosterone is added to the sample and allowed to dialyze across a membrane. Once equilibrium has been achieved, the solutions on both sides of the membrane are sampled to determine the percent free testosterone. Total testosterone is measured by HPLC with tandem mass spectrometry. Serum free testosterone is then calculated by multiplying the percent free testosterone by the total testosterone concentration with unit correction.

When elevated: Elevated free testosterone in women may suggest hyperandrogenism associated with PCOS, congenital adrenal hyperplasia, adrenal or ovarian androgen-secreting tumors, or exogenous androgen use. In men, elevated levels may indicate exogenous testosterone administration, anabolic steroid use, or, rarely, androgen-secreting neoplasms. Elevated free testosterone in the context of low SHBG (e.g., obesity, insulin resistance) may occur even when total testosterone appears normal. When low: Low free testosterone in men is consistent with hypogonadism and may be associated with symptoms including decreased libido, erectile dysfunction, fatigue, reduced muscle mass, increased adiposity, and mood disturbances. In women, low free testosterone may contribute to reduced libido, fatigue, and diminished sense of well-being, though clinical significance in females remains an area of ongoing research. Low free testosterone can occur with elevated SHBG (e.g., aging, hyperthyroidism, oral estrogen use) even when total testosterone is within the normal range.

Free testosterone is highly relevant to athletes because it directly influences muscle protein synthesis, recovery, strength gains, and training adaptation. Athletes often monitor this marker to assess whether training load, nutrition, and recovery are supporting healthy hormonal status, since chronic overtraining or inadequate recovery can suppress free testosterone and impair performance and adaptation.