This study aimed to investigate the effect of acute psychological stress on autonomic function and arterial stiffness, and to test a mediating role of changes in autonomic function between acute stress and arterial stiffness. Eighty-five healthy female adults were randomized into either an experimental or control group. The Trier Social Stress Test (TSST) was used to induce acute psychological stress. Autonomic function (measured by pre-ejection period [PEP] from cardiac impedance and high frequency [HF] of heart rate variability [HRV]) and arterial stiffness (measured by carotid and femoral pulse wave velocity [cfPWV] and augmentation index [AIx]) were assessed before and after the TSST. The mean age of the participants was 28.78 (±9.84) years old. Experimental group participants had a significant increase in cfPWV (p = .025) and AIx (p = .017) following the stressor, compared with those in the control group, after controlling for age, body mass index, and systolic blood pressure. However, no significant group differences were observed in changes in PEP (p = .181) and HF (p = .058). Changes in PEP and HF were neither associated with changes in cfPWV (p = .975 and p = .654, respectively), nor in AIx (p = .376 and p = .323, respectively). The results suggest that even a brief period of mild to moderate stress, which does not cause sustainable changes in autonomic function, may still exert significant adverse effects on arterial stiffness. The changes in arterial stiffness were not related to changes in autonomic function. Future experimental studies with several measurement points are recommended to identify distinct effects of stress on autonomic function and arterial stiffness.