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Separation from the group is a potent stressor for sheep and may have an adverse effect on animal welfare. Therefore, the present study was initiated to determine the effect of short-term seclusion of sheep on their welfare indicators in terms of behavior and endocrine level. Sixteen ewes aged 3-4 years were kept together for one month to form a group and get emotionally accustomed to each other. Thereafter, one individual ewe was daily separated from their group and kept in a separate shed to ward off their visual and tactile contact with other group mates. Thus, the 16 ewes of the group were separated from their group individually for 24 hours one after another for one time for each individual. It was found that the dry matter intake, water intake, and body weight were reduced significantly (P < 0.05) after isolation from the group. Respiration rate, pulse rate, and rectal temperature increased significantly (P < 0.05) after separation from the group. Cortisol, thyroxine, and triiodothyronine levels were elevated after separation from the pen mate. The total time spent feeding, ruminating, and lying decreased significantly (P < 005), whereas standing and location time increased significantly (P < 0.05) after separation from the group. The percentage of time spent on different behavior at different hours also differ significantly (P < 0.05) after separation from the group as compared to when the ewes were in the group. Therefore, it can be concluded that seclusion from the group elicits a significant stress on animals, which may be due to emotional distress.