Biphasic secretion is an autonomous feature of many endocrine micro-organs to fulfill physiological demands. The biphasic activity of islet β-cells maintains glucose homeostasis and is altered in type-2 diabetes. Nevertheless, underlying cellular or multicellular functional organizations are only partially understood. High-resolution non-invasive multi-electrode array recordings permit simultaneous analysis of recruitment, of single-cell and of coupling activity within entire islets in long-time experiments. Using this approach, we addressed the organizational modes of both, 1st and 2nd phase, in mouse and human islets under physiological and pathophysiological conditions. Our data provide a new uni- and multicellular model of islet β-cell activation: during the 1st phase, small but highly active β-cell clusters are dominant, whereas during the 2nd phase electrical coupling generates large functional clusters via multicellular slow potentials to favor an economic sustained activity. Post-prandial levels of glucagon-like peptide-1 (GLP-1) favor coupling only in the 2nd phase, whereas aging and glucotoxicity alter coupled activity in both phases. In summary, biphasic activity is encoded upstream of vesicle pools at the micro-organ level by multicellular electrical signals and their dynamic synchronization between β-cells. The profound alteration of the electrical organization of islets in pathophysiological conditions may contribute to functional deficits in type-2 diabetes.