OBJECTIVE: The goal of this study was to explore the development and implementation of a protocol for real-time fMRI neurofeedback (rtfMRI-nf) and to assess the potential for enhancing the selective brain activation using stimuli from Virtual Reality (VR). In this study we focused on two specific brain regions, supplementary motor area (SMA) and right inferior frontal gyrus (rIFG). Publications by other study groups have suggested impaired function in these specific brain regions in patients with the diagnoses Attention Deficit Hyperactivity Disorder (ADHD) and Tourette's Syndrome (TS). This study explored the development of a protocol to investigate if attention and contextual memory may be used to systematically strengthen the procedure of rtfMRI-nf. METHODS: We used open-science software and platforms for rtfMRI-nf and for developing a simulated repetition of the rtfMRI-nf brain training in VR. We conducted seven exploratory tests in which we updated the protocol at each step. During rtfMRI-nf, MRI images are analyzed live while a person is undergoing an MRI scan, and the results are simultaneously shown to the person in the MRI-scanner. By focusing the analysis on specific regions of the brain, this procedure can be used to help the person strengthen conscious control of these regions. The VR simulation of the same experience involved a walk through the hospital toward the MRI scanner where the training sessions were conducted, as well as a subsequent simulated repetition of the MRI training. The VR simulation was a 2D projection of the experience.The seven exploratory tests involved 19 volunteers. Through this exploration, methods for aiming within the brain (e.g. masks/algorithms for coordinate-system control) and calculations for the analyses (e.g. calculations based on connectivity versus activity) were updated by the project team throughout the project. The final procedure involved three initial rounds of rtfMRI-nf for learning brain strategies. Then, the volunteers were provided with VR headsets and given instructions for one week of use. Afterward, a new session with three rounds of rtfMRI-nf was conducted. RESULTS: Through our exploration of the indirect effect parameters - brain region activity (directed oxygenated blood flow), connectivity (degree of correlated activity in different regions), and neurofeedback score - the volunteers tended to increase activity in the reinforced brain regions through our seven tests. Updates of procedures and analyses were always conducted between pilots, and never within. The VR simulated repetition was tested in pilot 7, but the role of the VR contribution in this setting is unclear due to underpowered testing. CONCLUSION: This proof-of-concept protocol implies how rtfMRI-nf may be used to selectively train two brain regions (SMA and rIFG). The method may likely be adapted to train any given region in the brain, but readers are advised to update and adapt the procedure to experimental needs.