BACKGROUND AND PURPOSE: The link between brain iron deficiency and RLS is now well established. In a related observation, several conditions that can deplete iron stores have been linked to increased probability of RLS. Blood donation has been linked to iron deficiency. It has thus been hypothesized that donating blood may be a risk factor for developing RLS. PATIENTS AND METHODS: Two thousand and five UK blood donors, ranging from first-time donors to some who had donated more than 70 times, completed the validated Cambridge-Hopkins RLS questionnaire (CH-RLSq) following their donation session. The questionnaire included a set of questions designed to diagnose RLS. The donors' histories of blood donations were determined both from self-report and from the National Blood Service database. RESULTS: A number of statistical models were constructed to determine whether the probability of RLS diagnosis was related to the history of blood donations. Controlling for age and sex, no evidence was found to suggest that a greater number or frequency of blood donations increased the risk of RLS. Even amongst sub-groups especially vulnerable to iron depletion through blood donation, such as vegetarians or low weight individuals, no evidence for an increased risk of RLS could be found. CONCLUSIONS: We found no evidence that the frequency or number of blood donations up to the UK maximum of three times a year would increase the risk of RLS.