Pulsatility is a fundamental feature of pancreatic islets and a hallmark of hormone secretion. Isolated pancreatic islets endogenously generate rhythms in secretion, metabolic activity, and intracellular calcium ([Ca(2+)](i)) that are important to normal physiological function. Few studies have directly compared oscillatory and nonoscillatory islets to identify possible differences in function. We investigated the hypothesis that the loss of these oscillations is a leading indicator of islet dysfunction by comparing oscillatory and nonoscillatory mouse islets for multiple parameters of function. Nonoscillatory islets displayed elevated basal [Ca(2+)](i) and diminished [Ca(2+)](i) response and insulin secretory response to 3-28 mm glucose stimulation compared with oscillatory islets, suggesting diminished glucose sensitivity. We investigated several possible mechanisms to explain these differences. No differences were observed in mitochondrial membrane potential, estimated ATP-sensitive potassium channel and L-type calcium channel activity, or cell death rates. Nonoscillatory islets, however, showed a reduced response to the sarco(endo)plasmic reticulum calcium ATPase inhibitor thapsigargin, suggesting a disruption in calcium homeostasis in the endoplasmic reticulum (ER) compared with oscillatory islets. The diminished ER calcium homeostasis among nonoscillatory islets was also consistent with the higher cytosolic calcium levels observed in 3 mm glucose. Inducing mild damage with low-dose proinflammatory cytokines reduced islet oscillatory capacity and produced similar effects on glucose-stimulated [Ca(2+)](i), basal [Ca(2+)](i), and thapsigargin response observed among untreated nonoscillatory islets. Our data suggest the loss of oscillatory capacity may be an early indicator of diminished islet glucose sensitivity and ER dysfunction, suggesting targets to improve islet assessment.