Summary Fat necrosis is a common complication after autologous breast reconstruction using a free flap, which can influence reconstruction outcome and patient satisfaction. Intraoperative near-infrared fluorescence imaging using indocyanine green permits visualization of flap perfusion. The aim of this study was to assess the effect of near-infrared fluorescence on fat necrosis incidence in patients undergoing an autologous breast reconstruction with a deep inferior epigastric perforator flap and to propose a standard working protocol for this technique. In this prospective study, patients undergoing one-sided autologous breast reconstruction with a deep inferior epigastric perforator flap were included. The standard deep inferior epigastric perforator flap procedure was complemented with near-infrared fluorescence imaging using the fluorescent tracer indocyanine green to evaluate flap perfusion. This cohort was compared to a retrospective cohort of deep inferior epigastric perforator flap procedures without near-infrared fluorescence imaging. Patients and surgery characteristics, as well as postoperative complications, were registered and compared. In both cohorts 24 patients were included. No significant differences were present between patients and surgery characteristics, including no difference in duration of surgery. In the prospective and retrospective group the incidence of fat necrosis was 4.2% and 33%, respectively (p-value = 0.023). Moreover, microsurgeons were positive about using near-infrared fluorescence as it either provided additional information about perfusion or confirmed clinical assessment. Our pilot study showed a significant decrease of fat necrosis in patients undergoing an autologous breast reconstruction with a deep inferior epigastric perforator flap when near-infrared fluorescence imaging was used to asses flap perfusion. This study provides a standardized working protocol for near-infrared fluorescence imaging. In future, large multicenter studies should focus on quantification of near-infrared fluorescence imaging.