ImportanceSARS-CoV-2-specific antibodies in human milk might protect the breastfed infant against COVID-19. One of the factors that may influence human milk antibodies is psychological stress, which is suggested to be increased in lactating women during the COVID-19 pandemic.ObjectiveTo determine whether psychological stress is increased in lactating women during the COVID-19 pandemic, and if maternal stress is associated with the level of SARS-CoV-2-specific antibodies in human milk.DesignPopulation-based prospective cohort study.SettingData collection took place in the Netherlands between October 2020 and February 2021.ParticipantsLactating women living in the Netherlands were eligible to participate in this study. In total, 2310 women were included.ExposuresStress exposure during the COVID-19 pandemic was determined using the Perceived Stress Scale (PSS) questionnaire and maternal lifetime stress was determined by the Life Stressor Checklist – revised (LSC-r) questionnaire.Main Outcome(s) and Measure(s)Stress experience during the COVID-19 pandemic was compared with a pre-pandemic cohort. SARS-CoV-2-specific antibodies in human milk were measured using an Enzyme-Linked Immunosorbent Assay (ELISA) with the Spike protein of SARS-CoV-2. The association between maternal stress and human milk antibodies was determined using a multiple regression model.ResultsThe PSS score of lactating mothers was not increased during the pandemic compared to the PSS score in the prepandemic cohort. Six hundred ninety-one participants had SARS-CoV-2-specific antibodies and were included in the regression models to assess the association between maternal stress and human milk antibodies. No association was found between PSS scores and human milk antibodies. In contrast, the LSC-r score was negatively associated with SARS-CoV-2-specific IgA in human milk (β = 0.98, 95% CI: 0.96–0.997, p = 0.03).Conclusions and RelevanceOur results suggest that lactating women in the Netherlands did not experience higher stress levels during the COVID-19 pandemic. Breastfed infants of mothers with high chronic stress levels receive lower amounts of antibodies through human milk, which possibly makes them more vulnerable to respiratory infections. This emphasizes the importance of psychological wellbeing during lactation.