It is well known that moderate exercise is beneficial to health. However, the effects of exercise on subjective symptoms in relation to mood and autonomic nervous function have not yet been fully examined. The purpose of this study was to investigate the effects of daily walking on subjective symptoms as well as on mood and autonomic nervous function in people who take no medication but have some general physical complaints. We assessed their symptoms by the Cornell Medical Index (CMI), and mood states by a profile of mood states (POMS) and a frontal alpha laterality ratio. Autonomic nervous function was evaluated by a supine rest basal level, reactivity to orthostatic challenge (physiological stimulus) and to a self-programmed videogame (psychophysiological stimulus) of heart rate (HR), heart rate variability (HRV), baroreflex sensitivity and blood pressure (BP). Repeated measures analysis of variance showed no significant group (control and walking group) ×time (pre- and post- walking period) interaction of CMI scores. In contrast, the A-H sub-scale (anger and hostility) of POMS and basal HR significantly decreased after a 4-week walking period in a walking group compared to a control group. Negative mood score of POMS reduced, and basal high-frequency component of HRV and reactivity to orthostatic challenge of baroreflex sensitivity increased marginally significantly compared to the control group. Multiple regression analysis revealed a significant contribution of A-H to the physical score of CMI, which showed a marginally significant reduction after the experimental period in the walking group. These results suggest that daily walking can improve mood states and shift autonomic balance to parasympathetic predominance, and may consequently contribute to the reduction of subjective symptoms.