Chronic exercise exerts beneficial effects on cardiovascular functions with multiple mechanisms. This study examined whether chronic exercise improves aortic endothelial calcium (EC [Ca²⁺]_i) signalling or heat-shock protein 72 (HSP72) expression in rats under stress. Five-week-old male Wistar rats were randomly divided into control and exercise groups. Rats in the exercise group underwent 8 week exercise training either by the access to a running wheel or by running on a treadmill. At the end of exercise periods, rats were exposed to restraint stress for 30 min, rested for another 30 min, and killed by CO₂ inhalation. The vascular functions in dissected aortas were examined by monitoring the acetylcholine-evoked EC [Ca²⁺]_i using a tissue flow chamber mounted on an epifluorescence microscope with digital ratio imaging capability, and by monitoring the vascular HSP72 protein expression using Western blotting. Stress in rats induced elevated heart rate (567 ± 54 vs. 388 ± 15 beats/min, stress vs. control, p<0.05, n=4), blood pressure (124 ± 22 vs. 97 ± 10 mmHg, stress vs. control, p<0.05, n=4), and plasma corticosterone level (455 ± 40 vs. 90 ± 25 pg/ml, 30 min post-stress vs. control, p<0.05, n=6). It also suppressed EC [Ca²⁺]_i signalling (250 ± 20 vs. 730 ± 140 nM at 10^-6M acetylcholine, for stressed and unstressed control, respectively, p0.05, n=2). Eight week exercise training ameliorated stress-induced reduction of EC [Ca²⁺]_i signalling (405 ± 60 vs. 250 ± 20 nM at 10^-6M acetylcholine, for exercised/stressed and unexercised/stressed, respectively, p<0.05, n=4) and enhanced stress-induced aortic HSP72 protein expression (0.92 ± 0.14 vs. 0.67 ± 0.19 for exercised/stressed and unexercised/stressed, respectively, p<0.05, n=8). Taken together, stress induced vascular endothelial dysfunction and evoked defense mechanisms, while chronic exercise ameliorated stress-induced endothelial dysfunction and enhanced stress-evoked defense mechanisms.