Biological indicators for stress reactions are valuable markers in psychophysiological research and clinical practice. Since the release of salivary enzyme alpha-amylase was reported to react to physiological and psychological stressors, we set out to investigate human salivary alpha-amylase changes employing a reliable laboratory stress protocol to investigate the reactivity of salivary alpha-amylase to a brief period of psychosocial stress. In a within-subject repeated-measures design, 24 healthy adults were exposed to the TSST and a control condition on separate days with randomized sequence. Salivary alpha-amylase, salivary cortisol and heart rate were repeatedly measured before, during and after both conditions. Significant differences between psychosocial stress and the rest condition in alpha-amylase activity [F(3.74,86.06)=4.52; P=0.003], cortisol levels [F(4.21,88.32)=12.48; P<0.001] and heart rate [F(1,22)=81.15; P<0.001] were observed, with marked increases before and after stress. The data corroborate findings from other studies that showed increased levels of alpha-amylase before and after psychological stress. We discuss the role of salivary alpha-amylase as a promising candidate for a reliable, noninvasive marker of psychosocial stress.