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Most existing studies on high-speed rail (HSR) seating comfort are based on subjective surveys and pressure maps; there is no physiological reporting on muscle fatigue and stress accrual with longer travel periods. The assessment of dynamic sitting performance in different travel conditions is rather difficult due to the impossibility of the traditional laboratory-based investigations to reproduce real-life posture variations. To surmount these limitations, the present research compares business, first-class, and economy-class seats in a real-world HSR travelling scenario through the application of multi-modal ergonomic testing, which involves the use of electromyography (EMG), skin conductance response (SCR), and scenario-based behavioral testing. The 30 participants were divided into two seating classes and placed in four functional situations: entertaining, dining, working, and resting. Whereas SCR was used to monitor autonomic stress responses, EMG was used to record muscular activity in the shoulders, lumbar, and neck. The results indicate that although business-class chairs reduce lumbar strain, they do not eliminate the weariness of upper limbs and necks, especially in the reclining position. The first-class seats are maximum in terms of working postures, but they cannot offer flexible support for varied body proportions, which results in head and lumbar discomfort. Owing to their low level of adjustability, economy-class seats produce the largest amount of muscular tension, despite their high level of lumbar and neck support. This research provides an understanding of adjustable, ergonomically idealized seating for trains in future models and the significance of incorporating behavioral and physiological information into dynamic seat analysis.