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A key element in the development and innovation of future aviation concepts and systems is research flight simulation. Research flight simulation is applied when the performance and perception of human pilots is a key measure of the overall assessment. This paper will give an overview of the research simulation set-up of the National Aerospace Laboratory (NLR), Amsterdam, the Netherlands, which is used for the human-in-the-loop evaluation of future operational concepts. Special attention is given to the research topic of Airborne Separation Assurance; often referred to as Free Flight. The presented set-up has proven to be a flexible evaluation tool for assessing human-in-the-loop performance when operating in a simulated future autonomous aircraft environment.