Obesity is a well-established risk factor for many chronic diseases. Incomplete insight exists in the causal pathways responsible for obesity-related disorders and consequently, in the identification of obese individuals at risk of these disorders. The Netherlands Epidemiology of Obesity (NEO) study is designed for extensive phenotyping to investigate pathways that lead to obesity-related diseases. The NEO study is a population-based, prospective cohort study that includes 6,673 individuals aged 45–65 years, with an oversampling of individuals with overweight or obesity. At baseline, data on demography, lifestyle, and medical history have been collected by questionnaires. In addition, samples of 24-h urine, fasting and postprandial blood plasma and serum, and DNA were collected. Participants underwent an extensive physical examination, including anthropometry, electrocardiography, spirometry, and measurement of the carotid artery intima-media thickness by ultrasonography. In random subsamples of participants, magnetic resonance imaging of abdominal fat, pulse wave velocity of the aorta, heart, and brain, magnetic resonance spectroscopy of the liver, indirect calorimetry, dual-energy X-ray absorptiometry, or accelerometry measurements were performed. The collection of data started in September 2008 and completed at the end of September 2012. Participants are followed for the incidence of obesity-related diseases and mortality. The NEO study investigates pathways that lead to obesity-related diseases. A better understanding of the mechanisms underlying the development of disease in obesity may help to identify individuals who are susceptible to the detrimental metabolic, cardiovascular and other consequences of obesity and has implications for the development of prevention and treatment strategies.