Vitamin D Deficiency: Which Blood Tests Do You Need?

Vitamin D is made in the skin from sunlight and absorbed from a short list of foods and supplements, then converted in the liver to 25-hydroxyvitamin D — the storage form, with a half-life of two to three weeks — and in the kidneys to the active hormone, 1,25-dihydroxyvitamin D, which raises calcium absorption from the gut and keeps bone mineralized. Deficiency means the stores are too low to support that work: calcium absorption falls, parathyroid hormone rises to pull calcium from bone, and over months to years bone softens (osteomalacia in adults, rickets in children) and fractures more easily.

The test is 25-hydroxyvitamin D, because it reflects total supply from skin and diet and is tightly regulated neither up nor down. The active 1,25 form is the wrong test for deficiency: the kidneys hold it normal or even high while stores run out, and it is reserved for kidney disease, granulomatous conditions such as sarcoidosis, and unexplained high calcium.

The thresholds come from the Institute of Medicine and the NIH: below 12 ng/mL (30 nmol/L) is deficient, 12–19 ng/mL inadequate for bone health in most people, and 20 ng/mL (50 nmol/L) or above sufficient. The Endocrine Society's 2011 guideline preferred 30 ng/mL or more, a target many clinicians still use for people with osteoporosis, malabsorption or fractures. Above about 50 ng/mL there is no added benefit, and above 100–150 the risk of toxicity rises. Results are seasonal — lowest in late winter, highest in late summer — so a February result and an August result are not comparable.

25-hydroxyvitamin D. The diagnosis, the baseline for replacement and the check that replacement worked. Modern assays measure D2 and D3 together; the total is what matters.

Calcium, phosphorus and alkaline phosphatase (on a comprehensive metabolic panel). These read the consequences. Calcium is usually normal in deficiency because parathyroid hormone defends it; a low calcium means severe, long-standing deficiency (or another cause). A low phosphorus with a high alkaline phosphatase is the biochemical signature of osteomalacia — bone that is being remodeled but not mineralized. Albumin corrects the calcium, and creatinine and eGFR matter because the kidneys activate vitamin D and because kidney disease changes the targets.

Parathyroid hormone (PTH). Rises when vitamin D is low — secondary hyperparathyroidism — and falls back as stores are repleted. A raised PTH with a low vitamin D confirms that the deficiency is biologically meaningful; a raised PTH with a high calcium is primary hyperparathyroidism, a different diagnosis that vitamin D testing often uncovers by accident.

Magnesium. The enzymes that activate vitamin D need magnesium, and a low magnesium is one reason a level fails to rise on replacement. Celiac serology (tissue transglutaminase IgA with a total IgA) is checked when the deficiency is severe, unexplained by sun and diet, accompanied by iron or B12 deficiency, or does not correct. Vitamin B12 and folate travel with vitamin D in the fatigue work-up and in malabsorption, which is why the three are panelled together.

The 2024 Endocrine Society guideline recommends against routine screening of healthy adults, because the trials of supplementing people with normal levels showed no benefit; it recommends testing people in whom a low result changes management. That list is long enough:

• Symptoms — deep bone pain and tenderness (lower back, hips, pelvis, legs), proximal muscle weakness or difficulty rising from a chair, muscle aches, fractures from minor falls, and the fatigue work-up when the easier explanations have been excluded.
• Bone disease — osteoporosis, osteopenia, a fragility fracture, or before starting bisphosphonate or denosumab therapy (which can drop calcium if vitamin D is low).
• Malabsorption — celiac disease, Crohn's disease, bariatric surgery, pancreatic insufficiency, cystic fibrosis, cholestatic liver disease.
• Chronic kidney disease, hyperparathyroidism, an unexplained high or low calcium, high alkaline phosphatase or kidney stones.
• Medications — long-term glucocorticoids, anticonvulsants (phenytoin, phenobarbital, carbamazepine), rifampin, some HIV drugs, orlistat, cholestyramine.
• High risk with little sun — older adults who are housebound or in care, darker skin at northern latitudes, full covering clothing, obesity, pregnancy with risk factors, and exclusively breastfed infants not receiving drops.

Preparation. No fasting is needed for vitamin D itself (fast for the glucose if a metabolic panel is drawn with it). Do not stop supplements before a baseline unless you want to know your unsupplemented level; do note them. Stop biotin for 72 hours — it interferes with many vitamin D immunoassays. Remember the season when you read the result, and recheck at the same time of year when comparing.

Below 12 ng/mL with a normal calcium and PTH: deficiency, caught before it has done harm — replace and recheck. Below 12 with a raised PTH, a low phosphorus and a high alkaline phosphatase: deficiency that has reached the bone, with osteomalacia likely; replacement plus a bone density scan and a look for malabsorption. 12–19: inadequate — replacement for most people with symptoms, bone disease or risk factors; optional and lower-stakes in a healthy adult. 20–29: sufficient for bone health by the IOM standard; people with osteoporosis, malabsorption or fractures are often brought to 30 or more. Above 50: more than needed; above 100: check the calcium, stop supplements and recheck.

A level that fails to rise after 8–12 weeks of replacement means poor absorption (celiac disease, bariatric surgery, pancreatic insufficiency), a low magnesium, an interfering drug, a dose that is too low for body weight, or the supplement is not being taken. A low vitamin D with a high calcium is not a vitamin D problem at all — it is primary hyperparathyroidism or another cause of hypercalcemia until proven otherwise, and the PTH decides.

Order online, walk into any Quest or Labcorp location with your requisition — no appointment or doctor's visit needed — and results post to your account, most within one to two business days.

Take a low result to your clinician. Replacement is straightforward but not one-size-fits-all: the form (D3 or D2), the amount and the duration depend on how low the level is, body weight, absorption, kidney function and whether calcium needs attention at the same time; high-dose loading courses are prescription territory and are followed by a recheck. The retest is at 8–12 weeks, when the level has plateaued, then yearly once stable. Severe bone pain with difficulty walking, a fracture after a minor fall, or muscle cramps and tingling with a low calcium need prompt care rather than a routine follow-up.