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Background: In many countries out-of-hours care faces serious challenges, including shortage of general practitioners, a high workload, reduced motivation to work out of hours, and increased demand for out-of-hours care. One response to these challenges is the introduction of nurse practitioner as doctor substitutes, in order to maintain the (high) accessibility and safety of out of hours care. Although nurse practitioners have proven to provide equally safe and efficient care during daytime primary care, it is unclear whether substitution is effective and efficient in the more complex out of hours primary care. This study aims to assess the effects of substitution of care from general practitioners to nurse practitioners in an out of hours primary care setting., Design: A quasi experimental study is undertaken at one "general practitioner cooperative" to offer out-of-hours care for 304.000 people in the South East of the Netherlands. In the experimental condition patient care is provided by a team of one nurse practitioner and four general practitioners; where the nurse practitioner replaces one general practitioner during one day of the weekend from 10 am to 5 pm. In the control condition patient care is provided by a team of five general practitioners during the other day of the weekend, also from 10 am to 5 pm. The study period last 15 months, from April 2011 till July 2012., Methods: Data will be collected on number of different outcomes using a range of methods. Our primary outcome is substitution of care. This is calculated using the number and characteristics of patients that have a consultation at the GP cooperative. We compare the number of patients seen by both professionals, type of complaints, resource utilization (e.g. prescription, tests, investigations, referrals) and waiting times in the experimental condition and control condition. This data is derived from patient electronic medical records. Secondary outcomes are: patient satisfaction; general practitioners workload; quality and safety of care and barriers and facilitators., Discussion: The study will provide evidence whether substitution of care in out-of-hours setting is safe and efficient and give insight into barriers and facilitators related to the introduction of nurse practitioners in out-of-hours setting., Trial Registration: ClinicalTrials.gov ID NCT01388374.