Quick Guide

Vitamin D is a group of fat-soluble nutrients made in the body on exposure to sunlight and from certain foods (oily fish and egg yolk). There are 5 forms of vitamin D, of which only two are important to maintaining health: vitamin D2 (produced by plants) and vitamin D3 (produced with sunlight), both of which, when ingested, form vitamin D 25-hydroxy. The vitamin D 25-hydroxy test is the best indicator of how much vitamin D is stored in your body. It’s the. A blood sample is collected at a lab or patient service center, and results are ready in one to three business days. An at-home vitamin D test is also available if you prefer to collect a sample from home.

If you’ve been feeling unusually tired, having bone or muscle pain, susceptible to infections, or experiencing mood changes, a vitamin D deficiency may be the issue. You don’t need to fast before the draw.

If you take vitamin D supplements, especially high-dose D3, tell your provider before the test. Supplementation can affect your results. If your result comes back low, your provider will likely recheck levels eight to 12 weeks after you start treatment to confirm they’re rising.

About the Vitamin D 25-Hydroxy Test

Purpose of the test

This test measures your body’s total vitamin D levels. Decreased vitamin D levels may be due to poor diet, lack of adequate sunshine exposure, or certain other health conditions (cystic fibrosis, Crohn’s disease, or celiac disease) that prevent the proper absorption of vitamin D. Kidney and liver diseases can also negatively affect vitamin D metabolism.

Vitamin D plays a key role in how your body handles calcium and phosphorus, key elements that support immune function, muscle health, and nerve function. When levels stay too low for too long, the consequences may include bone loss, fractures, and immune problems. In rare cases, elevated vitamin D levels (due to taking too many over-the-counter supplements, but not excessive sun exposure) can result in vitamin D toxicity. Elevated vitamin D can also alter calcium and phosphorus levels, resulting in complications with the kidneys, intestines, and bones. According to the NIH Office of Dietary Supplements, the 25-OH vitamin D test is the best indicator of vitamin D status and is the standard measure used in research and clinical practice.

  • Screening: Routine screening for vitamin D levels is not done. Checks vitamin D in people at higher risk of deficiency: older adults, exclusively breast-fed infants, those with limited sun exposure, people with dark skin, obese people, people who have had gastric bypass surgery, and people with malabsorption conditions.
  • Diagnosis: Ordered when symptoms like bone pain, fatigue, muscle weakness, or frequent infections point to vitamin D deficiency.
  • Monitoring: Tracks whether vitamin D levels are rising after supplementation or dietary changes, typically rechecked eight to 12 weeks into treatment.

The vitamin D 25-hydroxy test is the standard screening test for vitamin D. Another test, the 1,25-dihydroxyvitamin D test (calcitriol), is reserved for specific conditions. 1,25-dihydroxy vitamin D is the most active form of vitamin D and is often decreased in chronic renal failure and hypoparathyroidism. Elevated levels may be seen in hyperparathyroidism and certain situations of decreased calcium or vitamin D intake. This test doesn’t detect milk allergy, celiac disease, or other causes of malabsorption. It only measures your vitamin D level.

What does the Vitamin D 25-Hydroxy Test measure?

Labs use two main methods to measure vitamin D 25-hydroxy in your blood.

  • Immunoassay: The most common method, measuring total 25-hydroxy vitamin D (D2 plus D3 combined).
  • LC-MS/MS (liquid chromatography-tandem mass spectrometry): A more precise method that can quantify D2 and D3 separately.

Some lab reports show D2 and D3 values separately alongside the total 25-OH vitamin D. The total is what your provider uses to assess your status. Sub-fraction values show whether your vitamin D is coming mainly from supplements (D2 or D3) or sun exposure (D3 only).

When should I get a Vitamin D 25-Hydroxy Test?

Consider testing if any of these apply:

  • Bone pain, fatigue, or muscle weakness that won’t go away
  • Frequent infections or slow recovery from illness
  • A condition that affects nutrient absorption (Crohn’s disease, celiac disease, cystic fibrosis, or liver disease)
  • A history of gastric bypass or other GI surgery
  • Limited sun exposure, darker skin, or age over 50
  • Obesity or a vegan diet with little vitamin D from food
  • A provider recommendation to check or monitor levels

No major U.S. health authority recommends universal vitamin D screening for all adults. The NIH Office of Dietary Supplements identifies specific groups who may benefit from testing or supplementation:

  • Adults over 75 years of age, who may benefit from empiric supplementation
  • People with malabsorption conditions (Crohn’s disease, celiac disease, cystic fibrosis, liver disease, or gastric bypass history)
  • People with obesity, since vitamin D is stored in fat tissue, and may be less available
  • Exclusively breastfed infants, who may not get enough vitamin D from breast milk alone (provider-ordered)
  • Anyone taking high-dose vitamin D supplements to watch for toxicity

The Endocrine Society’s 2024 clinical practice guideline on vitamin D similarly does not recommend routine population-wide screening but supports targeted testing in higher-risk groups.

Vitamin D levels are naturally lower in late winter and higher in late summer. A borderline result in February may look different in August, so keep that in mind if you’re close to a cutoff.

How It Works

How to get tested

This test is ordered through a healthcare provider, clinic, or hospital lab. Testing.com connects users with CLIA-certified laboratory partners, including LabCorp and Quest Diagnostics, for in-lab blood draws. Results come back through a secure online account or patient portal within one to three business days.

No special prep is needed beyond telling your provider about any vitamin D supplements you take. The blood draw takes a few minutes.

Before the test

You don’t need to fast. That’s different from glucose or lipid panels, which do require fasting. The confusion comes up because vitamin D is sometimes ordered as part of a broader panel that includes fasting tests. If you’re getting only the vitamin D 25-hydroxy test, eat and drink normally.

Tell your provider about all vitamin D supplements you take before the draw. If you took a high-dose D3 supplement the morning of your test, let the lab know. It can briefly raise your result.

Several medications can lower vitamin D levels. Tell your provider if you take any of these:

  • Corticosteroids (such as prednisone)
  • Anticonvulsants (such as phenytoin or phenobarbital)
  • Rifampin (an antibiotic used for tuberculosis)
  • Orlistat (a weight-loss medication)
  • Cholestyramine (a cholesterol-lowering medication)

Certain medications, including certain corticosteroids, weight-loss drugs, anticonvulsants, heart medications, blood pressure medicines, laxatives, aluminum-based products, and cholesterol-lowering medicines, can interfere with vitamin D metabolism and affect test results. No dietary restrictions, activity limits, or other prep steps are needed.

During the test

  1. Check in at the patient service center and confirm your name and date of birth.
  2. A technician draws blood from a vein in your arm. The draw takes a few minutes.
  3. The technician places a small bandage over the site. Keep it on for about 15 minutes to ensure bleeding has stopped. You can eat, drink, and go about your day right away.

You’ll feel a brief pinch when the needle goes in. Some people notice minor bruising afterward. That’s normal. The whole visit takes about 10 to 15 minutes. Call your provider if you have lasting pain, swelling, or signs of infection (redness, warmth, or discharge) at the draw site. These are uncommon.

After the test

Results are typically ready within one to three business days after the lab receives your sample, though timing can vary by lab.

You’ll get an email when the results are ready. View them in your secure online account. If your test was ordered through a provider’s office, results may come through their patient portal on a slightly different timeline.

What do my results mean?

Your result is reported in nanograms per milliliter (ng/mL). The ranges below are based on NIH Office of Dietary Supplements guidance reflecting the National Academies of Sciences, Engineering, and Medicine (NASEM) thresholds.

A few things can affect how your result reads. Lab methods can vary from lab to lab: immunoassay and LC-MS/MS can produce slightly different numbers for the same sample. If your result is borderline, ask which method your lab used. Season matters too. Levels are naturally lower in late winter and higher in late summer, so a borderline low in February may look different by August.

If your report shows D2 and D3 separately, the total 25-OH vitamin D is the number your provider uses.

If your results are sufficient (20 ng/mL or greater)

Your body has enough vitamin D for bone health, immune function, and calcium regulation. More is not necessarily better. Levels above 50 ng/mL may be linked to potential adverse effects.

If you were being monitored after starting supplementation, a sufficient result means it’s working. No changes are needed unless your provider says otherwise. If symptoms like fatigue or bone pain continue despite a sufficient level, follow up with your provider for further investigation.

If your results are low (less than 20 ng/mL)

Low results are common and treatable. Vitamin D deficiency is considered when levels fall below 20 ng/mL, and at risk of inadequacy (vitamin D insufficiency) when levels fall between 20 -30 ng/mL. Both may need treatment, but the approach can differ.

Providers typically recommend vitamin D3 supplements, more dietary sources (fatty fish, fortified milk, egg yolks), and safe sun exposure. Plan to retest eight to 12 weeks after starting supplementation to confirm your levels are rising.

If your level is very low (below 20 ng/mL), your provider may recommend higher-dose prescription supplementation. If vitamin D levels do not rise with treatment, follow-up testing may be needed to check for malabsorption conditions like celiac disease, Crohn’s disease, or liver or kidney disease.

A borderline low result in winter may reflect normal seasonal variation. If you don’t have symptoms and your result is close to 20 ng/mL, your provider may suggest retesting in late summer before starting supplements.

If your results are high (greater than 50 ng/mL)

Sun exposure alone generally does not cause this. Your skin self-regulates and typically stops making vitamin D when levels are sufficient. Toxicity almost always comes from taking too much vitamin D in supplement form, often high-dose D3 without monitoring.

Symptoms include nausea, vomiting, weakness, constipation, poor appetite, stomach pain, and weight loss. Left untreated, high vitamin D may raise blood calcium (hypercalcemia), which can cause confusion, kidney damage, and heart rhythm problems. The Cleveland Clinic notes that vitamin D toxicity is almost always the result of taking excessive supplement doses rather than diet or sun exposure.

Stop vitamin D supplementation and contact your provider. Follow-up testing often includes a calcium test and a kidney function panel. Don’t try to manage a high result on your own.

FAQs

What is the difference between the 25-hydroxy and 1,25-dihydroxy vitamin D tests?

The 25-hydroxy vitamin D test (this test) measures inactive, stored vitamin D and is the standard way to check your overall vitamin D status. The 1,25-dihydroxy vitamin D test measures the active hormone-like form (calcitriol) and is used for specific conditions like chronic kidney disease and granulomatous disorders (such as sarcoidosis). Most people need the 25-hydroxy test. If you aren’t sure which one was ordered, ask your provider.

How do I increase my vitamin D levels if my result is low?

Vitamin D3 supplements are the most reliable way to raise levels. Food sources include fatty fish (salmon, tuna, mackerel), egg yolks, beef liver, vitamin D-fortified milk, orange juice, and cereals. Other common nutritional deficiencies include Vitamin B12, iron, and iodine. Each can be tested to ensure adequate levels. Safe sun exposure helps too, though the amount you need varies by skin tone, season, and geography. Talk with your provider before starting high-dose supplementation, and plan to retest in eight to 12 weeks.

Can children get a vitamin D 25-hydroxy test?

Yes. Vitamin D deficiency in children can cause rickets, a condition where bones don’t mineralize properly. Provider-ordered testing is the right route for children under 18. Some consumer lab services allow testing for children 10 and older with parental consent, but a pediatrician should guide interpretation and treatment.

Is vitamin D toxicity possible from sun exposure?

Sun exposure alone generally does not cause vitamin D toxicity. Your skin typically self-regulates vitamin D production and stops when levels are sufficient. Toxicity almost always comes from taking too much vitamin D in supplement form. If you take high-dose D3 supplements, periodic testing is the best way to keep your levels in a safe range.

Why does my lab report show vitamin D2 and D3 separately?

Some labs use a specific technique (LC-MS/MS), which can quantify vitamin D2 (from plant and fungal sources) and D3 (from sunlight and animal sources), separately. Both are added together to give your total 25-OH vitamin D. That total is the number your provider uses. If your report shows only a total, your lab used an immunoassay method.

Can vitamin D levels vary by season?

Yes. Vitamin D levels are naturally lower in late winter and early spring and higher in late summer, because sunlight drives vitamin D production in your skin. If your result is borderline low and you tested in winter, your provider may recommend retesting in summer before starting supplementation, or may treat based on your symptoms and risk factors.

Related Tests

Sources

See More