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Background Adolescents who engage in nonsuicidal self-injury (NSSI) show a high sensitivity to ostracism, and its effects may be amplified by dysregulated stress-system function. However, ecologically grounded experimental studies examining multilevel stress responses in adolescent mental health remain scarce. We used a novel, ecologically valid in-person ostracism paradigm to assess subjective, autonomic, and endocrine responses in adolescents with recent NSSI and to characterize stress trajectories. Methods Fifty adolescents with recent NSSI (mean age = 16.40 years, 76% female) were randomized (1:1) to live social inclusion or exclusion in a face-to-face ball-toss task based on the Cyberball paradigm. Salivary cortisol was sampled at five points from pre-task to 40 min post-task. Heart rate (HR) and heart rate variability (HRV) were recorded at baseline and during the interaction. Subjective stress, tension, and NSSI urges were assessed before and after the task. Perceived exclusion and psychological need threat were also assessed. Results We found clear ostracism effects on perceived exclusion and basic psychological need threat. Cortisol declined across the paradigm in both conditions (inclusion/exclusion), but the decline was flatter after exclusion, indicating attenuated hypothalamic-pituitary-adrenal axis downregulation. HR decreased modestly over time across conditions, and HRV diverged by condition: it decreased during exclusion and increased during inclusion. There were no condition-related changes in self-reported stress, tension, or NSSI urges. Conclusions These findings suggest a decoupling between subjective and psychobiological stress systems in adolescents with NSSI in response to ostracism, characterized by rapid parasympathetic withdrawal and a blunted HPA axis downregulation. This pattern may reflect heightened physiological sensitivity to social threat despite limited conscious distress. Trial registration: This study has been registered at the German Clinical Trials Register (ID: DRKS00025905, https://drks.de/search/en/trial/DRKS00025905).