Sleep loss and psychosocial stress often co-occur in today’s society, but there is limited knowledge on the combined effects. Therefore, this experimental study investigated whether one night of sleep deprivation affects the response to a psychosocial challenge. A second aim was to examine if older adults, who may be less affected by both sleep deprivation and stress, react differently than young adults. 124 young (18–30 years) and 94 older (60–72 years) healthy adults participated in one of four conditions: i. normal night sleep & Placebo-Trier Social Stress Test (TSST), ii. normal night sleep & Trier Social Stress Test, iii. sleep deprivation & Placebo-TSST, iv. sleep deprivation & TSST. Subjective stress ratings, heart rate variability (HRV), salivary alpha amylase (sAA) and cortisol were measured throughout the protocol. At the baseline pre-stress measurement, salivary cortisol and subjective stress values were higher in sleep deprived than in rested participants. However, the reactivity to and recovery from the TSST was not significantly different after sleep deprivation for any of the outcome measures. Older adults showed higher subjective stress, higher sAA and lower HRV at baseline, indicating increased basal autonomic activity. Cortisol trajectories and HRV slightly differed in older adults compared with younger adults (regardless of the TSST). Moreover, age did not moderate the effect of sleep deprivation. Taken together, the results show increased stress levels after sleep deprivation, but do not confirm the assumption that one night of sleep deprivation increases the responsivity to an acute psychosocial challenge.