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Objective In order to characterize the feature of stress response induced by stressor with both physical and psychological natures, the effect of water restriction performed in different experimental modes on the physiological parameters, psychological behavioral manifestations and brain c-Fos expressions were observed and compared. Methods 58 male Wistar rats were used and randomly divided into three experimental groups (n= 18 for each) and a control group (n=4). In control group, the rats were allowed to access drinking water freely at all experimental period. In the experimental groups the water supply to the rats was restricted. In timed water supply (TW) group, the water was supplied twice a day,10 min for each in fixed hours every day. In empty bottle-served (EB) and water-restricted (WR) groups, the water was served only once a day for 10 min, either in the early morning or evening, and in the other time point scheduled for water supply only an empty bottle without water was provided in the EB group and nothing was given in the WR group. The quantities of drank water and eaten food, weight-gaining, and behavior score were observed every day. The serum level of corticosterone was assayed and the rats were sacrificed with fixative perfusion of 3 d, 7 d or 14 d respectively, following water restriction (n=6 for each time point in each group). The brain c-Fos expressions were examined with immunohistochemistry. Results The slowing down of weight-gaining, rise of serum corticosterone level, occurrence of psychological behavioral manifestations of unpeaceful restlessness such as exploring and attacking, enhance of c-Fos expression in the subfornical organ (SFO), median preoptic nucleus (MnPO), area postrema (AP), hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON), medial (MeA) and central (CeA) amygdaloid nucleus and ventrolateral septum (LSV) were noticed in both EB and WR groups, except the nucleus of solitary tract (NTS) in which the Fos expression was decreased. The changes of Fos expression in most of nuclei in EB group began at day 3, at least persisted till day 7, and backed down at day 14. While in WR group, similar changes started at day 7 and reached its peak at day 14. In TW group, only the concentration of corticosterone at day 7 was slightly increased and the rest indexes observed were unchanged. Conclusion The results indicate that water restriction induces physical as well as psychological stress responses. And the water restrictions experimentally executed in different modes result in different manifestations of behavioral response and brain immediately early gene expression in discrete brain nuclei/regions.