The COVID-19 pandemic led to increased internalizing symptoms (IS) among adults, with notable interindividual differences. Cross-sectional studies suggest that both biological (physiological stress) and psychological (socio-emotional) factors independently contribute to IS. This longitudinal study examined whether physiological stress during the pandemic moderated the relationship between socio-emotional vulnerability and IS. In June 2020, 94 adults (average age 34.16, SD = 8.86) provided a 6 cm hair sample, allowing the quantification of cumulative hair cortisol concentrations (HCC) during the three months preceding the pandemic (Segment A) and the three months of the first wave of the pandemic in Quebec, Canada (Segment B). We calculated HCC reactivity (rHCC) as the percent change between the two segments. A socio-emotional composite score (SECS) was derived using the Anxiety Sensitivity Index, Intolerance of Uncertainty Scale, and Perseverative Thinking Questionnaire. Every three months from June 2020 to March 2021, participants completed the Depression Anxiety Stress Scales and Impact of Event Scale. Analyses revealed an interaction between Time, SECS, and rHCC on stress, and between SECS and rHCC on depressive symptoms. High rHCC was associated with a stronger correlation between SECS and stress symptoms in September and December 2020 (these months were significant transition periods during the pandemic in Quebec). Additionally, individuals with low rHCC exhibited greater depressive symptoms, regardless of time. Our findings suggest that the activation of the physiological stress system at the beginning of the pandemic was associated with increased stress symptoms during subsequent transition periods. Conversely, lower activation of the stress system contributed to greater depressive symptoms over time, particularly in individuals with heightened socio-emotional vulnerability. This highlights the role of physiological responses in modulating emotional outcomes under prolonged stress and adds to the growing literature on vulnerability factors in response to enduring stressors.