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       Objective To analyze the characteristics and trends of physiological and biomechanical signal and to explore relative factors resulting in fatigue in walking with load. Methods Cardio-pulmonary function parameters, shoulder force, trunk pressure, and perceived fatigue were sampled simultaneously in six healthy men during 30-minute simulated walking (speed at 5km/h, slope at 0%) experiment under five different conditions of backpack loads (25kg，29kg，34kg，37kg，39kg). Results Heart rate (HR), breathing rate (BR), minute ventilation volume (VE), oxygen consumption (VO2) gradually increased as the payload increased, and the increasing rate became bigger during 37-39kg trials. Shoulder force was high correlated with payload. Pressures at shoulder, waist, and back regions were influenced by the tension degree of waist and chest belts, and 37kg was the turning point. The scores of perceived fatigues in shoulder and whole body were higher than those of back and waist. Conclusions Thirty-seven kilogram is the threshold value of fatigue in cardio-pulmonary function, and cardio-pulmonary exercising capacity should be considered when we study simulated walking with payloads greater than 37kg. Shoulder force and pressure are considered as main factors for fatigue evaluation.