Abstract Background Behavioral performance of health professionals is essential for adequate patient care. There is little information on behavioral skills acquisition after simulation training of neonatal resuscitation. To assess the behavioral skills of fellows in neonatology before and after a simulation training program on neonatal resuscitation. Methods From March/2019 to February/2020, a prospective cohort with 12 second-year fellows in neonatology were evaluated during three training cycles (16 hours each) in manikin-based simulation of neonatal resuscitation with standardized scenarios. Each cycle lasted one month, followed by a 3-months interval. One video-recorded scenario of about 10 minutes was performed for each fellow at the beginning and at the end of each training cycle. Therefore, each fellow was recorded 6 times: before and after each one of three training cycles. Anxiety of the fellows was assessed by the Beck Anxiety Inventory applied before the first training cycle. The videos were independently analyzed in a random order by three trained facilitators using the Behavioral Assessment Tool. The behavioral performance was evaluated by repeated measures analysis of variance adjusted for anxiety and for previous experience in neonatal resuscitation. Results Fellows` overall behavioral performance improved comparing the moment before the first training and after the second training. The specific skills - communication with the team, delegation of tasks, allocation of attention, use of information, use of resources and professional posture - showed a significant improvement after the second month of training. No further gains were noted with the third training cycle. Anxiety was observed in 42% of the fellows and its presence worsened their behavioral performance. Conclusion An improvement in behavioral performance was observed comparing the moment before the first training and after the second training, without further gains after the third training. It is worth noting the important role of anxiety as a modulator of acquisition and retention of behavioral skills in health professionals in training.