Heart rate variability (HRV) is one of the frequently used biomarkers of autonomic activation. In the presented psychophysiological research, we observe HRV during stress in highly anxious and allergic people. In both groups, current research documented the dysregulation of stress response on neuroendocrine level, which may also appear in the cardiovascular level represented by the heart rate (HR) and heart rate variability (HRV). The situation of acute stress was created using the PSST (psychosocial stress test), which simulates a public speech task before the committee combined with general knowledge and arithmetic tasks. In a study of 27 subjects, we compared time-domain and frequency-domain aspects of HRV during relaxation and PSST, in context of trait anxiety measured by STAI, and the incidence of allergic disease. In data analysis we found significant differences in HRV between phases of PSST, which supports the claim that PSST is a sufficient stress stimulus. Subjects with allergy and high anxious subjects had lower overall HRV, which corresponds to prior findings.