Background <p>Reusable flexible ureteroscopes (rfURS) are costly and fragile equipment that can be easily damaged during surgery. We aimed to investigate the change in the surgeon’s reaction time during retrograde intrarenal surgery (RIRS), which may be a risk factor for rfURS damage in prolonged cases.</p> Methods <p>The patients who underwent RIRS with rfURS by the same surgeon were prospectively included in the study and the collected data were retrospectively analyzed. A phone application program (Reflex Test 2.2.4) designed to evaluate the user’s reaction time with visual color change was used to evaluate the reaction time. According to the application format, the surgeon had to press the square when the rectangle’s color changed from red to blue. The surgeon’s reaction time was evaluated just before the operation and then every 10&#xa0;minutes of the surgery.</p> Results <p>A total of 109 patients were included in the study. The mean stone volume was 1050.4 ± 874.5 mm³ (62.9-3889.7). The procedure time increased significantly as the stone volume increased. The mean reaction time of the pre-procedure was 0.330 ± 0.059&#xa0;seconds (s). 0,338 ± 0.053&#xa0;s (10th min.), 0.342 + 0.057&#xa0;s (20th min), 0.356 ± 0.061&#xa0;s (30th min), 0.361 ± 0.047&#xa0;s (40th min), 0.364 ± 0.042&#xa0;s (50th min), 0.376 ± 0.046&#xa0;s (60th min) and 0.384 ± 0.032&#xa0;s (more than 70&#xa0;min) for 10, 20, 30, 40, 50, 60 and more than 70&#xa0;min, respectively. The sequential reaction times of the procedure demonstrated a significant difference (<i>p</i> &lt; 0.001).</p> Conclusion <p>Compared to the pre-procedure period, the surgeon’s reaction time increased during the surgery. This result may indicate increased surgeon fatigue during extended procedures, and additional investigation of its possible clinical implications is needed.</p>