Salivary cortisol measurement has proved useful for the non-invasive study of the hypothalamic-pituitary-adrenocortical axis, and salivary alpha-amylase has been suggested as a comparable marker for the sympathetic system. Despite some studies showing an increase in salivary alpha-amylase after challenges that stimulate the sympathetic nervous system, questions remain about interpretation. The aims of this study were to explore the stability of salivary alpha-amylase, its diurnal profile, response to the cold hand test, and correlation with cortisol. Salivary alpha-amylase was stable following 5 days at room temperature, and five freeze-thaw cycles. Its diurnal profile was opposite to that of cortisol. There was no salivary alpha-amylase response to the cold hand stress test, in the morning (11am) or afternoon (3pm), unlike cortisol which showed a response in the afternoon in the same samples. There was no correlation between salivary alpha-amylase and cortisol at any time. In conclusion, salivary alpha-amylase is stable to a range of conditions. Its diurnal pattern is compatible with sympathetic stimulation. Lack of response to the cold hand test suggests that secretion of salivary alpha-amylase is controlled by mechanisms more complex than sympathetic regulation alone.