Abstract: Systems and methods for avoiding problems due to over-pressurization in a closed microfluidic device when the contained fluid is heated, by providing a well-defined volume for expansion of the liquid contents during heating, such as in one embodiment filling the expansion volume with air, and in other embodiments, filling the expansion volume with vapor-phase fluid from the liquid in the microfluidic device before the device is sealed and in yet other embodiments providing structures to maintain uniform temperatures over the entire closed volume in the microfluidic device.