The goal of this observational study is to collect fecal and serum samples from patients with restless legs syndrome (RLS) and healthy controls for differential microbiota analysis to identify key bacterial species involved in the pathogenesis of RLS. Subsequently, metabolomic analysis will be performed on fecal and serum samples from RLS patients to identify key regulatory molecules in iron metabolism disorder pathways. Finally, correlation analysis will be used to determine which specific bacterial species and metabolites are involved in the pathogenesis of RLS. The main question it aims to answer is: i) Do RLS patients exhibit gut microbiota dysbiosis compared to healthy controls? ii) Which specific bacterial species and metabolites are linked to iron metabolism abnormalities and the pathogenesis of RLS?

Inclusion Criteria:

• ≥18 years old;
• The diagnostic criteria all meet the five diagnostic criteria for restless legs syndrome established by the International Committee for the Study of Restless Legs Syndrome (IRLSSG) (2014 revised version)

Exclusion Criteria:

• Suffering from serious physical illnesses, such as tumors, cardiovascular diseases, diabetes, stroke, etc.;
• Drug abuse, alcoholism, acute poisoning;
• Antibiotic use within the 4 weeks preceding sampling;
• Arobiotics or prebiotics use within the 4 weeks preceding sampling;
• Iron supplements use within the 4 weeks preceding sampling.

We will recruit 50 patients with restless legs syndrome from among those attending the Sleep Medicine Center of Chongqing Traditional Chinese Medicine Hospital during the period December 2025 to December 2026.