AbstractAutism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder influenced by both genetic and environmental factors. Recently, gut dysbiosis has emerged as a powerful contributor to ASD symptoms. In this study, we recruited over 100 age-matched sibling pairs (between 2 and 8 years old) where one had an Autism ASD diagnosis and the other was developing typically (TD) (432 samples total). We collected stool samples over four weeks, tracked over 100 lifestyle and dietary variables, and surveyed behavior measures related to ASD symptoms. We identified 117 amplicon sequencing variants (ASVs) that were significantly different in abundance between sibling pairs across all three timepoints, 11 of which were supported by at least two contrast methods. We additionally identified dietary and lifestyle variables that differ significantly between cohorts, and further linked those variables to the ASVs they statistically relate to. Overall, dietary and lifestyle features were explanatory of ASD phenotype using logistic regression, however, global compositional microbiome features were not. Leveraging our longitudinal behavior questionnaires, we additionally identified 11 ASVs associated with changes in reported anxiety over time within and across all individuals. Lastly, we find that overall microbiome composition (beta-diversity) is associated with specific ASD-related behavioral characteristics.