During type 1 diabetes (T1D) progression, beta cells become dysfunctional and exhibit reduced first-phase insulin release. While this period of beta cell dysfunction is well established, its cause and underlying mechanism remain unknown. To address this knowledge gap, live human pancreas tissue slices were prepared from autoantibody- negative organ donors without diabetes (ND), donors positive for one or more islet autoantibodies (AAb+), and donors with T1D within 0-4 years of diagnosis (T1D+). Dynamic imaging and physiological secretion studies were conducted to assess the extent and impact of T cell infiltration on beta cell function through immunolabeling, Ca 2+ imaging, and perifusion assays for insulin secretion. Beta cells were identified in living slices by ENTPD3 cell surface staining. Beta cells from both ND and AAb+ donors exhibited normal cytosolic Ca 2+ mobilization in response to high glucose and KCl. Beta cells from donors with T1D had significantly diminished Ca 2+ responses to high glucose but preserved responses to KCl. In T1D, glucose responsiveness was impaired in both T cell-infiltrated and non-infiltrated insulin positive islets, supporting the concept that beta cell dysfunction is independent of close spatial association between beta cells and T cells. Fixed tissue staining and gene expression profiling of laser-capture microdissected islets revealed significant decreases in markers of glucose metabolism to ATP in beta cells from donors with T1D but no changes in endoplasmic reticulum (ER) stress markers. From these data, we posit that functional defects occur in beta cells during T1D pathogenesis independent of local T cell infiltration and beta cell destruction. Additionally, these beta cell metabolic defects contribute to the clinical manifestation of dysglycemia at T1D diagnosis despite a remaining mass of beta cells.