Vitamin D, 25-Hydroxy, Total (Immunoassay) Test
The Vitamin D, 25-Hydroxy, Total test measures circulating 25-hydroxyvitamin D (D2 and D3 combined) in serum, the recommended marker of vitamin D status. It is used to diagnose deficiency and insufficiency, guide and monitor supplementation, and assess bone and mineral disorders.
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Biomarkers
Blood (serum)
Sample
2–4 business days
Turnaround
No — however, practitioners should document recent vitamin D supplementation and product type (D2 vs D3), as both affect interpretation
Fasting
What is the Vitamin D, 25-OH test?
The Vitamin D, 25-Hydroxy, Total Test measures circulating 25-hydroxyvitamin D — the combined D2 and D3 forms — which is the recommended marker of vitamin D status. Because of its long half-life and stability, it provides an integrated picture of vitamin D supply from sun exposure, diet, and supplementation, and is the standard test for diagnosing deficiency. Within the assessment of bone and mineral health, 25-OH vitamin D is the first-line test, while the active metabolite calcitriol is reserved for specific disorders of vitamin D metabolism rather than status assessment. The total assay is preferred over D3-only measurement because it captures D2 from supplements and fortified sources. This immunoassay-based method is widely used and clinically validated for routine status assessment; LC-MS/MS methods may be selected when precise D2/D3 differentiation or resolution of assay interference is required. Practitioners order it to investigate musculoskeletal symptoms, malabsorption, and metabolic bone disease, and to guide repletion.
What does the Vitamin D, 25-OH test measure?
25-Hydroxyvitamin D is the major circulating form of vitamin D and the recommended marker of vitamin D status. It is generated by hepatic 25-hydroxylation of vitamin D3 (cholecalciferol, from cutaneous synthesis and animal sources) and vitamin D2 (ergocalciferol, from plant and supplement sources). It circulates bound predominantly to vitamin D-binding protein with a half-life of approximately two to three weeks, making it a stable integrated measure of supply. It is the substrate for renal 1-alpha-hydroxylation to the active hormone calcitriol.
Total 25-OH vitamin D, capturing both D2 and D3, is the standard index of vitamin D adequacy and reflects combined dietary, supplemental, and sun-derived sources. It is used to diagnose deficiency and insufficiency, guide and monitor supplementation, and investigate disorders of bone and calcium metabolism. The active metabolite calcitriol (1,25-OH vitamin D) is not used to assess status because its short half-life and tight regulation make it an unreliable measure of stores.
Why is the Vitamin D, 25-OH test ordered?
- Investigation of suspected vitamin D deficiency — bone pain, proximal myopathy, unexplained musculoskeletal symptoms, or fatigue
- Assessment and monitoring of metabolic bone disease, including osteoporosis, osteomalacia, and before initiating antiresorptive therapy
- Evaluation of patients with malabsorptive conditions — coeliac disease, inflammatory bowel disease, or post-bariatric surgery — at high risk of deficiency
- Monitoring of vitamin D repletion in patients on supplementation, to confirm adequacy and avoid excess
- Workup of disordered calcium and parathyroid metabolism, interpreted alongside calcium, phosphate, and PTH
- Assessment in patients with chronic kidney or liver disease where vitamin D activation or hydroxylation may be impaired
- Evaluation of at-risk populations — limited sun exposure, darker skin pigmentation, older adults, or institutionalised patients — as part of preventive assessment
Sample collection and turnaround
Sample type
Blood (serum)
Fasting required
No — however, practitioners should document recent vitamin D supplementation and product type (D2 vs D3), as both affect interpretation
Collection method
Venipuncture at patient service centre
Turnaround
2–4 business days
Collection notes
Document supplementation dose, timing, and form. Advise patients to withhold high-dose biotin supplementation for 48–72 hours prior to collection to avoid immunoassay interference.
Specimen requirements
Minimum 0.5 mL serum. Separate serum from cells per Quest protocol. Specimen stable refrigerated; protect from prolonged light exposure and follow Quest transport guidelines.
What can affect Vitamin D, 25-OH results?
Supplementation raises 25-OH vitamin D over weeks. Because the marker integrates supply over its long half-life, recent dosing and product (D2 vs D3) should be documented when interpreting status.
Some immunoassays under-recover or over-recover the D2 form relative to D3, so total values in patients on ergocalciferol can misrepresent true status. LC-MS/MS resolves D2 and D3 separately.
Vitamin D is sequestered in adipose tissue, lowering circulating 25-OH vitamin D for a given intake. Higher supplementation doses are often required to achieve sufficiency.
Fat malabsorption from coeliac disease, inflammatory bowel disease, or bariatric surgery impairs absorption of fat-soluble vitamin D, lowering 25-OH vitamin D independent of intake.
Altered binding-protein concentrations in pregnancy and some illnesses shift total 25-OH vitamin D without necessarily changing bioavailable hormone, complicating interpretation.
Biotin can interfere with streptavidin-biotin immunoassay platforms, producing falsely altered results. Patients should withhold high-dose biotin for 48–72 hours before collection.
What do Vitamin D, 25-OH results mean?
Elevated
Values above 100 ng/mL raise concern for vitamin D toxicity, typically from excessive supplementation, and may be accompanied by hypercalcaemia. Toxicity from sun exposure does not occur. Supplementation history should be reviewed and calcium checked where excess is suspected.
Normal
Values of 30–100 ng/mL indicate sufficiency under the Endocrine Society framework. Note that the Institute of Medicine considers 20 ng/mL adequate for most of the population, so the chosen threshold affects classification.
Reduced
Values below 20 ng/mL indicate deficiency and below 30 ng/mL insufficiency under Endocrine Society thresholds. Deficiency warrants assessment of dietary intake, sun exposure, malabsorption, and adiposity, and prompts repletion with follow-up monitoring.
Note: The sufficiency threshold is genuinely contested between guideline bodies, so deficiency status depends on the cut-off applied. Total assays may handle D2 imperfectly, and binding-protein changes can shift total values without altering bioavailable hormone. 25-OH vitamin D — not the active calcitriol — is the correct marker of status.
Frequently asked questions
25-OH vitamin D has a long half-life and stable concentration, giving an integrated measure of supply from sun, diet, and supplements. The active form, calcitriol, is tightly regulated and short-lived, making it unreliable for assessing stores.
How to access the Vitamin D, 25-OH test
The Vitamin D, 25-OH 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
- Holick MF, et al. Evaluation, Treatment, and Prevention of Vitamin D Deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. (2011)
- Ross AC, et al. The 2011 Report on Dietary Reference Intakes for Calcium and Vitamin D from the Institute of Medicine. J Clin Endocrinol Metab. (2011)
- Sempos CT, et al. Vitamin D assays and the definition of hypovitaminosis D. Br J Clin Pharmacol. (2018)
- Bouillon R, et al. Skeletal and Extraskeletal Actions of Vitamin D: Current Evidence and Outstanding Questions. Endocr Rev. (2019)
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.