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This study was conducted to determine whether an increase in salivary free cortisol would be reliably elicited by a midday meal, thus providing a convenient physiological challenge to the hypothalamic-pituitary-adrenal (HPA) axis, and whether this cortisol release depended on the protein content of the meal.In healthy men, free cortisol was measured in saliva samples taken before and after two identical protein-rich midday meals (39% energy as protein) and compared with a day on which no meal was eaten. Next, in healthy women in a nonclinical setting, salivary cortisol was measured before and after a protein-rich meal (32% energy as protein) on one day and a low-protein meal (5% energy as protein) on another day. Measures of mood, appetite, and psychological well-being were also taken.An acute meal-dependent increase in salivary cortisol occurred, which was reliable over 2 test days. This increase in cortisol depended on the proportion of protein in the meal, increasing after the high-protein but not the low-protein meal. The extent of this increase in cortisol correlated significantly with poor psychological well-being in women. Some postmeal improvement of mood (positive affect) was associated with the high- but not the low-protein meal.The cortisol response to meals may have implications for the effects of meal composition on mood, cognitive function, and food choice. The measurement of free cortisol in saliva provides a psychologically stress-free and reliable technique to assess the cortisol response to a standard protein-rich meal, ie, a physiological challenge to the HPA axis in men and women that could be investigated in naturalistic settings outside the laboratory.