In healthy, non-challenged individuals, the secretion of cortisol typically follows a diurnal profile characterized by a peak in the period following waking (cortisol awakening response) and a gradual decline throughout the day. In addition, cortisol secretion is increased in response to acutely stressful stimuli, particularly stressors involving social evaluation. The current study is the first to assess the impact of an anticipated acute laboratory stressor upon the typical diurnal pattern of HPA activation and relationship to acute cortisol secretion. A sample of 23 healthy young adults provided salivary cortisol samples at four time points (immediately upon awakening, 30-min post-awakening, 1200 h and before bed) on 2 consecutive days. On the second day, participants attended the laboratory and undertook an anticipated acute socially evaluative stressor immediately following provision of their 1200 h saliva sample. Heart rate, blood pressure and mood were recorded immediately before and after the stressor and at 10 and 20 min post-stressor along with additional salivary cortisol samples. Typical patterns of cortisol secretion were observed on both days and exposure to the laboratory stressor was associated with the expected increases in cortisol, heart rate, blood pressure and negative mood. However, significant differences in diurnal cortisol secretion were observed between the two days with greater secretion, in particular, during the period following awakening, evident on the day of the anticipated laboratory stressor. Furthermore, secretion of cortisol during the period following awakening was positively related to secretion during the acute reactivity periods. This is the first study to integrate a laboratory stressor into a typical day and assess its impact on indices of diurnal cortisol secretion in an ambulatory setting. The current findings support the notion that the cortisol awakening response is associated with anticipation of the upcoming day and the subsequent demands required of the individual.