Physiological monitoring during actual flight operations remains an underexplored area in aviation human factors research. This study investigated real-time autonomic responses, heart rate (HR), heart rate variability (HRV), and respiratory rate (RR) in pilots of varying experience levels during Cessna 172 flights, including in-flight emergency events. Unlike simulator-based investigations, this work introduced a novel, field-based approach using continuous ECG-based monitoring to capture vital parameters under operational load. Flight scenarios comprised baseline and routine operations (REST), a nominal visual-flight circuit (NOR), and controlled engine-failure events (EF) executed at take-off, downwind, and final approach. Comparative analysis revealed that the cadet pilot exhibited a 21.53% HR increase and 18.13% HRV decline during emergencies, while the experienced pilot showed smaller shifts (19.31% and 15.81%, respectively). ANOVA results supported these differences with higher F-values in cadet responses. These findings underscore the physiological variability introduced by inexperience and support the integration of in-flight vital-parameter monitoring into adaptive pilot training.