Asthma–chronic obstructive pulmonary disease (COPD) overlap syndrome (ACOS) seems an important clinical phenotype, but multiple definitions have been proposed. This study's objectives were to assess the effect of different ACOS definitions on prevalence, patient characteristics and exacerbations.5675 individuals aged 45–65 years, with 846 asthma/COPD patients, were included in the Netherlands Epidemiology of Obesity study between 2008 and 2012, and followed-up for a median of 1.8 years. ACOS was defined by recent consensus criteria and five other definitions, based on registry, questionnaires and lung function.Prevalence of ACOS in the asthma/COPD population ranged between 4.4% and 38.3%, depending on the definition used. Agreement between registry-based and self-reported ACOS was 0.04 and 0.41 when lung function (forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) <0.7) was added. With registry or self-report defined ACOS, only 51% and 33% had FEV1/FVC <0.7. Patient characteristics were similar, but asthma duration was longer with self-reported compared with registry-based ACOS (mean difference 22 years (95% CI 12–33)). Exacerbation risk was highest with registry-based ACOS compared with asthma (adjusted incidence rate ratio 1.6 (95% CI 1.2–2.1)).This study adds important knowledge about agreement between ACOS definitions and their relation with exacerbations. Given the low agreement, differences in prevalence, patient characteristics and risk of exacerbations, consensus about ACOS definition in different care settings is urgently needed.