Laboratory Testing for Suspected Restless Legs Syndrome
In a patient with suspected restless legs syndrome, order serum ferritin, transferrin saturation (drawn in the morning after avoiding iron supplements for 24 hours), complete blood count, electrolytes with renal function (creatinine/eGFR), and consider TSH, HbA1c, and calcium to identify secondary causes. 1, 2
Essential Iron Studies (Highest Priority)
Serum ferritin and transferrin saturation are mandatory in all patients with suspected RLS and must be drawn under specific conditions to ensure accuracy 1, 2:
- Draw blood in the morning after the patient has avoided all iron-containing supplements and foods for at least 24 hours 1, 2
- Ferritin has diurnal variation and recent iron intake falsely elevates results 1
- Inflammation can raise ferritin independent of true iron stores, which is why transferrin saturation is also required 1
- RLS-specific thresholds differ from general population: supplement if ferritin ≤75 ng/mL or transferrin saturation <20% 1, 2
The American Academy of Sleep Medicine emphasizes that both parameters are necessary because ferritin alone can be falsely elevated by inflammation, while transferrin saturation <20% indicates functional iron deficiency even when ferritin appears adequate 1.
Complete Blood Count
A CBC is reasonable to obtain to assess for iron-deficiency anemia, which is a well-established secondary cause of RLS with significantly higher prevalence in affected patients 1. This identifies overt anemia that may require more aggressive iron repletion, particularly in patients with known iron deficiency 1. Women are at much higher risk for iron deficiency without anemia (IDNA), with a relative risk of 5.51 3.
Renal Function Testing
Electrolytes and renal function (creatinine/eGFR) are essential to identify chronic kidney disease, which is a secondary cause of RLS with increasing prevalence as kidney function declines 1, 4. End-stage renal disease is a major secondary cause of RLS and requires different treatment algorithms 4. Stage 4 CKD (eGFR 15-29 mL/min, not on dialysis) is strongly associated with RLS independent of iron status 5.
Additional Metabolic Screening
The following tests help identify other secondary causes 1:
- TSH: Part of standard endocrine screening for RLS 1
- HbA1c: Screens for diabetes mellitus, which is associated with RLS and peripheral neuropathy that can mimic RLS 1
- Calcium: Included in baseline metabolic assessment 1
- Urine albumin-to-creatinine ratio: Assesses for renal disease 1
Critical Testing Pitfalls to Avoid
Do not order polysomnography or periodic limb movement studies as part of routine RLS diagnosis—these should be reserved for cases where the diagnosis is uncertain or when assessing treatment response in research settings 1. The diagnosis of RLS is clinical, based on four essential criteria (urge to move legs, symptoms worse with rest, relief with movement, circadian pattern) 1, 6.
Do not use general population iron thresholds—ferritin <70 ng/mL is the best cutoff for identifying possible iron deficiency in RLS patients with inflammatory conditions 5. The standard cutoff of 45-50 ng/mL is inadequate in patients with acute and chronic inflammatory conditions 5.
Always measure transferrin saturation before initiating iron therapy to exclude hemochromatosis and iron overload 7. Patients with elevated pre-treatment transferrin saturation or serum ferritin should undergo evaluation to determine the cause before iron treatment 7.
Clinical Context
A thorough neurologic examination is essential to identify conditions like peripheral neuropathy, radiculopathy, or arthritis that may present with leg discomfort but are not RLS 1. Without proper differential diagnosis, 16% of patients who do not have RLS will be misclassified if clinicians only assess symptom criteria without excluding mimics 1. Blood work helps distinguish primary RLS from secondary causes: renal function identifies uremia-related RLS, HbA1c identifies diabetic neuropathy, and iron studies guide treatment 1.