Introduction: Individuals with hearing disorders such as tinnitus often experience high levels of stress, anxiety, depression, and decreased sound tolerance. These factors may be related to maladaptive autonomic regulation, affecting their response to acute stress. Heart rate variability (HRV) is a reliable measure of autonomic activity, but its interaction with psychological factors is not well understood. Objectives: The aim was to develop a multimodal stress induction protocol in a non-clinical population and to determine how psychological factors influence physiological responses. Methods: Electrocardiograms were recorded from 30 healthy young adults during three stress tasks: mental arithmetic, noise exposure, and a cold pressor test. Heart rate (HR) and root mean square of successive differences (RMSSD) were used to index autonomic activity. Participants also completed questionnaires on perceived stress, noise sensitivity, anxiety, and depression to examine relationships with HRV. Results: All tasks produced significant time-dependent effects on HRV features, but to different extents. Mental arithmetic produced the largest increase in HR and decrease in RMSSD. In contrast, the noise task showed a significant decrease in HR without a change in RMSSD, suggesting that the task induced relaxation. In the CPT, females showed increased HR but a decrease in RMSSD, whereas males showed the reverse pattern. Psychological factors significantly interacted with HRV dynamic changes during mental and noise tasks, but not during CPT. Conclusions: HRV responses to acute stress are non-stationary and influenced by psychological factors. Thus, this protocol presents an original approach to study autonomic activity in stress-related conditions such as tinnitus.