This study explores a new class of duplex microfluidic device which utilizes a dual perifusion network to simultaneously perform live-cell optical imaging of physiological activities and study insulin release kinetics on two islet populations. This device also incorporates on-chip staggered herringbone mixers (SHMs) to increase mixing efficiency and facilitate the generation of user-defined chemical gradients. Mouse islets are used to simultaneously measure dynamic insulin release, changes in mitochondrial potentials, and calcium influx in response to insulin secretagogues (glucose and tolbutamide), and show a high signal-to-noise ratio and spatiotemporal resolution of all measured parameters for both perifusion chambers. This system has many potential applications for studying β-cell physiology and pathophysiology, as well as for therapeutic drug screening. This dual perifusion device is not limited to islet studies and could easily be applied to other tissues and cells without major modifications.