Mental stress triggers positive inotropic and chronotropic effects as well as peripheral vasoconstriction. This alters the pulse arrival time (PAT), the duration between electrical excitation of the ventricles and arrival of the pulse wave in the periphery. We conducted a study to examine PAT during five rest blocks and under mental stress utilizing the Mannheim Multicomponent Stress Test. Electrocardiograms as well as finger and earlobe photoplethysmograms were recorded. PAT was calculated for over 135,000 heartbeats from 42 healthy volunteers as the time duration between the R peak in the electrocardiogram and the following pulse onset in the respective photoplethysmogram. To identify the effect of mental stress, block-wise PAT means were statistically analyzed with repeated measures ANOVA. The analyses showed significant differences between the block means for both PAT measures (p < 0.001). Post-hoc tests revealed significantly reduced PAT during the stress block compared to all rest blocks for both PAT measures (p < 0.001). We found no significant differences between the rest blocks. Our results support that PAT is a sensitive vital parameter for the detection of mental stress in healthy volunteers. This holds true for both measurement positions, the finger and the earlobe.