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Summary form only given. We examined how long-term walking affects gait and physiological rhythm. Twelve normal subjects performed three hours above-ground walking at their own preferable pace. Subjects were divided into two groups according to whether the mean gait cycle time in the second-half increased (group A) or decreased (group B) from that in the first-half. Group A subjects showed significantly higher subjective level of fatigue than Group B subjects. Mean power frequency (MPF) of EMG recorded from tibialis anterior significantly decreased with time, indicating that it can also be used to evaluate muscle fatigue induced by weak dynamical movement such as walking. In group A subjects, moreover, mean value and coefficient of variance of gait cycle time increased significantly with time, and largest Lyapunov exponent of back-waist acceleration in vertical direction decreased significantly with time. During the rest period before the walking task, mean RR-interval in group A subjects was significantly shorter than that in group B subjects. From these results, and taking the timing of each index when it reached the significant level into account, we proposed a hypothetical model of gait and physiological responses to physical fatigue.