In healthy individuals, plasma insulin levels oscillate in both fasting and fed states. Numerous studies of isolated pancreata and pancreatic islets support the hypothesis that insulin oscillations arise because the underlying rate of insulin secretion also oscillates; yet, insulin secretion has never been observed to oscillate in individual pancreatic β-cells. Using expressed fluorescent vesicle cargo proteins and total internal reflection fluorescence (TIRF) microscopy, we demonstrate that glucose stimulates human pancreatic β-cells to secrete insulin vesicles in short, coordinated bursts of ∼70 vesicles each. Randomization tests and spectral analysis confirmed that the temporal patterns of secretion were not random, instead exhibiting alternating periods of secretion and rest, recurring with statistically significant periods of 15–45 s. Although fluorescent vesicles arrived at the plasma membrane before, during, and after stimulation, their rate of arrival was significantly slower than their rate of secretion, so that their density near the plasma membrane dropped significantly during the cell's response. To study in greater detail the vesicle dynamics during cyclical bursts of secretion, we applied trains of depolarizations once a minute and performed simultaneous membrane capacitance measurements and TIRF imaging. Surprisingly, young fluorescent insulin vesicles contributed at least half of the vesicles secreted in response to a first train, even though young vesicles were vastly outnumbered by older, nonfluorescent vesicles. For subsequent trains, young insulin vesicles contributed progressively less to total secretion, whereas capacitance measurements revealed that total stimulated secretion did not decrease. These results suggest that in human pancreatic β-cells, young vesicles are secreted first, and only then are older vesicles recruited for secretion.