•PMDD women showed marked cycle-related variation in subjective stress reactivity.•Notably, high arousal negative affect toward stress arose in the late luteal phase.•PMDD was linked to a delayed CAR peak and a flattened daily cortisol slope.•High negative and low positive affect predicted high cortisol output across groups.•PMDD women showed reduced cortisol reactivity toward rumination. Premenstrual Dysphoric Disorder (PMDD) is characterized by significant emotional, physical and behavioral distress during the late luteal phase that remits after menses onset. Outlined as a new diagnostic category in DSM-5, the mechanisms underlying PMDD are still insufficiently known. Previous research suggests that PMDD exacerbates with stressful events, indicating a dysregulation of the hypothalamic-pituitary-adrenal axis. However, studies measuring stress-related processes in affected women in real-time and real-life are lacking. We conducted an Ambulatory Assessment (AA) study to compare subjective stress reactivity together with basal and stress-reactive cortisol activity across the menstrual cycle in women with and without PMDD. Women with current PMDD (n = 61) and age- and education matched controls (n = 61) reported momentary mood, rumination, and daily events via smartphones at semi-random time points 8 times a day over two consecutive days per cycle phase (menstrual, follicular, ovulatory, and late luteal). Twenty minutes after assessments participants collected saliva cortisol samples. Three additional morning samples determined the cortisol awakening response (CAR). Women with PMDD reported particular high daily life stress and high arousal negative affect (NAhigh) towards stressors during the late luteal phase. High momentary stress levels were linked to lower levels of high arousal positive affect (PAhigh) and to higher levels of rumination in PMDD women compared to controls irrespective of cycle phase. Across groups, more stress was linked to higher levels of low arousal NA (NAlow) and to lower levels of low arousal PA (PAlow). Moreover, PMDD was associated with a delayed CAR peak and a flattened diurnal cortisol slope. While neither group showed cortisol reactivity towards daily life stress directly, high momentary NAhigh and low momentary PA predicted high levels of cortisol across groups, whereas high momentary rumination predicted high cortisol output only in healthy women. In this AA-study we identified important stress-related psychological and endocrinological within-person variability in women with PMDD during daily life. Further research is warranted targeting identified AA-based mechanisms to study their predictive role for the clinical course of PMDD and to provide evidence-based therapeutic options for affected women.