One increasingly popular type of alternative therapy is aromatherapy, but scientific validation in this field is still rare. The aim of this study was to investigate the effect of rose oil (Rosa damascena Mill, Rosaceae) on human autonomic parameters and emotional responses in healthy subjects after transdermal absorption. In order to exclude any olfactory stimulation the inhalation of the fragrances was prevented by breathing masks. Forty healthy volunteers participated in the experiments. Five autonomic parameters, i.e. blood pressure, breathing rate, blood oxygen saturation, pulse rate, and skin temperature, were recorded. Emotional responses were assessed by means of rating scales. Compared to placebo, rose oil caused significant decreases of breathing rate, blood oxygen saturation and systolic blood pressure, which indicate a decrease of autonomic arousal. At the emotional level, subjects in the rose oil group rated themselves as more calm, more relaxed and less alert than subjects in the control group. These findings are likely to represent a relaxing effect of the rose oil and provide some evidence for the use of rose oil in aromatherapy, such as causing relief of depression and stress in humans.