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Background: Chronic stress is an important risk factor in the development of obesity. While research suggests chronic stress is linked to excess weight gain in children, the biological or behavioral mechanisms are poorly understood., Objective: The objectives of the Family Stress Study are to examine behavioral and biological pathways through which chronic stress exposure (including stress from COVID-19) may be associated with adiposity in young children, and to determine if factors such as child sex, caregiver-child relationship quality, caregiver education, and caregiver self-regulation moderate the association between chronic stress and child adiposity., Methods: The Family Stress Study is a prospective cohort study of families recruited from 2 Canadian sites: the University of Guelph in Guelph, Ontario, and McMaster University in Hamilton, Ontario. Participants will be observed for 2 years and were eligible to participate if they had at least one child (aged 2-6 years) and no plans to move from the area within the next 3 years. Study questionnaires and measures were completed remotely at baseline and will be assessed using the same methods at 1- and 2-year follow-ups. At each time point, caregivers measure and report their child's height, weight, and waist circumference, collect a hair sample for cortisol analysis, and fit their child with an activity monitor to assess the child's physical activity and sleep. Caregivers also complete a web-based health and behaviors survey with questions about family demographics, family stress, their own weight-related behaviors, and their child's mental health, as well as a 1-day dietary assessment for their child., Results: Enrollment for this study was completed in December 2021. The final second-year follow-up was completed in April 2024. This study's sample includes 359 families (359 children, 359 female caregivers, and 179 male caregivers). The children's mean (SD) age is 3.9 years (1.2 years) and 51% (n=182) are female. Approximately 74% (n=263) of children and 80% (n=431) of caregivers identify as White. Approximately 34% (n=184) of caregivers have a college diploma or less and nearly 93% (n=499) are married or cohabiting with a partner. Nearly half (n=172, 47%) of the families have an annual household income ≥CAD $100,000 (an average exchange rate of 1 CAD=0.737626 USD applies). Data cleaning and analysis are ongoing as of manuscript publication., Conclusions: Despite public health restrictions from COVID-19, the Family Stress Study was successful in recruiting and using remote data collection to successfully engage families in this study. The results from this study will help identify the direction and relative contributions of the biological and behavioral pathways linking chronic stress and adiposity. These findings will aid in the development of effective interventions designed to modify these pathways and reduce obesity risk in children., Trial Registration: ClinicalTrials.gov NCT05534711; https://clinicaltrials.gov/study/NCT05534711., International Registered Report Identifier (irrid): DERR1-10.2196/48549., (©Shannon M Pare, Elizabeth Gunn, Katherine M Morrison, Alison L Miller, Alison M Duncan, Andrea C Buchholz, David W L Ma, Paul F Tremblay, Lori Ann Vallis, Nicola J Mercer, Jess Haines. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 20.06.2024.)