Background <p>Camouflaging autistic traits is suggested to increase stress and the risk of autistic burnout. However, the relationship with psychological and biological markers of stress and the influence of familial factors on this relationship remain unclear.</p> Methods <p>In a neurodiverse twin sample (<i>N</i> = 315; 69 diagnosed with autism), we examined associations between camouflaging behaviors (operationalized as the discrepancy between the Autism Diagnostic Observation Schedule 2nd edition [ADOS-2] and the Autism Quotient [AQ]), rated stress-related symptoms, and biological long-term stress measured via hair cortisol concentration (HCC). Associations were analyzed across the full sample, accounting for age, sex, and HCC confounders, as well as within-twin pairs, implicitly adjusting for genetic and environmental confounding.</p> Results <p>Across individuals, camouflaging was associated with increased HCC, particularly in autistic and adult subsamples, while associations with stress-related symptoms were only evident in adults. Within-pair analyses revealed no associations in the full sample, suggesting familial confounding. Interestingly, in adjusted within-pair models in autistic and adult twin-pairs, camouflaging was linked to <i>lower</i> HCC.</p> Limitations <p>Only the adult participants provided self-reports of autistic traits (AQ) and stress-related symptoms, while parent-reports were used for the younger participants. This difference in reporting sources may have reduced the accuracy of data for the younger subgroups in our sample.</p> Conclusions <p>The findings indicate that camouflaging is associated with increased biological long-term stress at the population level but that familial factors may influence this relationship. Future research is needed to explore these complex dynamics and their implications for mental health and adaptive functioning in autistic individuals.</p>