The current study investigated autonomic arousal regulation and habituation effects in adults with autism (n = 29) compared to adults without autism (n = 29), across multiple measurement moments in an experimental setting. Based on the Neurovisceral Integration Model, which explains hyperarousal to be underlying to the difficulties in autonomic regulation and reduced habituation to the environment, heart rate variability (HRV) and respiratory sinus arrhythmia (RSA) were monitored through photoplethysmography (PPG) during three fMRI sessions. Two domains measuring stress and anxiety of the Amsterdam Resting State Questionnaire (ARSQ) served as a behavioural measure of atypical autonomic arousal, as hyperarousal leads to elevated stress and anxiety levels. Statistical analyses revealed no significant group differences in RSA or HRV, neither within the first fMRI scanning session participants encountered, nor across three scanning sessions, suggesting similar patterns of habituation between groups in terms of autonomic arousal. Contrary to expectations, both groups showed increasing autonomic arousal across sessions. However, self-reported stress and anxiety revealed a significant group difference in habituation across sessions: while both groups reported decreasing stress and anxiety levels over time, autistic individuals reported higher levels overall and adjusted more quickly. The null effect findings for HRV and RSA align with previous research reporting no significant differences in autonomic regulation in autism. Additionally, the discrepancy between the autonomic arousal results and self-reports align with multiple studies finding no links between symptoms of anxiety and HRV. In conclusion, the fMRI environment itself may not produce differences in autonomic arousal between groups, supporting the notion that fMRI may serve as a neutral and non-threatening environment for autistic people. Furthermore, it is important to recognize that self-reported stress and anxiety are distinct constructs, and the absence of autonomic differences does not rule out the possibility of having other self-reported emotional experiences. Interpretations of these findings are limited by modest sample size, missing data during the initial adjustment phase in the scanner and the absence of respiratory recordings.  Overall, this study provide a clearer understanding of autonomic regulation in autism, suggesting that reduced habituation may not be a universal mechanism in autistic people, but instead might be influenced by contextual factors and individual variability.