Adequate recovery and habituation to acute stressors in daily life are essential for mental health. One potential moderator might be the use of different emotion regulation (ER) strategies contributing to interindividual differences in vulnerability to chronic stress. Rumination has been linked to impaired endocrine adaptation, whereas reappraisal tendencies have been associated with boosted habituation to psychosocial stress. Yet, no experimental study has directly compared the causal effects of these strategies on psychoneuroendocrine responses to repeated stress. To address this gap, 91 healthy participants (47 women) underwent a short Trier Social Stress Test (TSST) twice on two consecutive days and were randomly assigned to a rumination, reappraisal, or control intervention in between. Cognitive-affective ratings indexed psychological stress, while salivary cortisol, alpha-amylase (sAA), and heart rate (HR) served as biomarkers. Across the entire sample, reduced increases in negative affect and cortisol in response to the second TSST confirmed successful habituation. As expected, rumination immediately increased negative affect, reduced positive affect, and lowered perceived coping abilities indicating successful induction of ruminative thinking. Moreover, it prevented physiological habituation to repeated stress, evidenced by stable cortisol and HR responses. Unexpectedly, reappraisal also lowered perceived coping abilities in men, followed by prolonged sAA reactivity to the first stress exposure, hinting at a sex-specific impairing effect of reappraisal on noradrenergic recovery. However, reappraisal neither affected cortisol recovery nor habituation, which may result from generally poor reappraisal performance. Together, these findings provide initial evidence for differential effects of rumination and reappraisal on psychophysiological adaptations to repeated TSST exposure.