Objective To observe the change of heart rate variability (HRV) in rats with bundling or isoflurane anesthesia and determine the differences of weight, pain threshold, and hormone levels of the HPA axis between rats with bundling and isoflurane anesthesia for 9 days (30 min/ day). Methods Male SD rats were randomly divided into three groups: a control group, bundling group, and isoflurane anesthesia group. By recording an electrocardiogram (ECG) for 15min, heart rate and HRV were analyzed in the three groups in an acute experiment. Body weight, pain threshold, and levels of CRH and ACTH (in serum, hypothalamus, and adrenal glands) were investigated after the 9?day intervention (30min/ day). Results 1) Compared with the control group, the heart rates of the bundling and isoflurane anesthesia groups increased significantly (both P < 0. 01). The heart rate of the bundling group also increased compared with that of the isoflurane anesthesia group ( P < 0. 05). Moreover, compared with the control and bundling groups, SDRR and HF values of the isoflurane anesthesia group were significantly decreased ( P < 0. 01, P < 0. 05) and LF and LF/ HF values were significantly increased ( P < 0. 05, P < 0. 01). 2) After the 9?day intervention, compared with that in the control group, the weight gains of the bundling and isoflurane anesthesia groups were decreased ( P < 0. 05). The weight gain of the bundling group was also decreased compared with that of the isoflurane anesthesia group ( P < 0. 05). 3) Compared with those in the control and isoflurane anesthesia groups, the pain threshold in the bundling group was decreased ( P < 0. 05), while there was no significant difference between the control group and the isoflurane anesthesia group. 4) Compared with the levels in the control and isoflurane anesthesia groups, the levels of corticotropin releasing hormone (CRH) in the hypothalamus and corticosterone (CORT) in the adrenal gland were decreased ( P < 0. 05). There was no significant change in the levels of CRH and CORT in serum ( P > 0. 05). Conclusions The low concentration of isoflurane caused an increase of heart rate and a decrease of heart rate variability in rats. It result ed in change in the functional status of the autonomic nervous system, which mainly involved sympathetic excitement. Compared with the control group, the 9?day bundling intervention caused chronic stress in rats. Isoflurane anesthesia is a method of reducing stress in rats during long?term interventions.