Abstract: Background: In Belgium, different routine surveillance systems are in place to follow-up Lyme borreliosis trends. However, accurate data on the disease and monetary burden for the different clinical manifestations are lacking. Despite recommended antibiotic treatment, a proportion of Lyme patients report persisting aspecific symptoms for six months or more (e.g. fatigue, widespread musculoskeletal pain, cognitive difficulties), a syndrome now named "post-treatment Lyme disease syndrome" (PTLDS). Controversy exists on the cause, incidence and severity of PTLDS. This study aims to estimate the incidence of PTLDS in patients with Lyme borreliosis and to quantify the disease burden and economic costs associated with the different clinical manifestations of Lyme borreliosis in Belgium. Methods: The project is a prospective cohort study in which about 600 patients with an erythema migrans and 100 patients with disseminated Lyme borreliosis will be followed up. Questionnaires, including the SF-36 vitality and pain subscale, the Cognitive Failure Questionnaire and the EQ-5D-5L, will be used to collect information on acute and persisting symptoms and the impact on quality of life. Symptom frequency and severity will be compared with self-reported pre-Lyme health status, a control group and existing Belgian population norms. Additionally, information on the associated costs and possible risk factors for the development of PTLDS will be collected. Discussion: A study of the health burden will allow evaluation of the relative importance of Lyme borreliosis in Belgium and information on the economic cost will help to formulate cost-effective measures. There are only few prospective studies conducted estimating the incidence of PTLDS and even though discussion exists about the prevalence of subjective symptoms in the general population, a control group of non-Lyme borreliosis participants has often not been included.