Subjects were wiped and cleaned with alcohol swap and DI water at area of interest before each sweat collection began. For whole-body and regional sweat analysis, regional sweat was collected by attaching our sweat patches (without impedance-based sweat rate sensor) at locations of interest. Subjects were asked to bike for 6 to 13 min with 4-min break in between for removing sweat patches, cleaning, weighing nude whole-body weight, and reattaching new sweat patches. Whole-body fluid loss rate was estimated from total nude body weight loss at each time interval. Each trial lasts for a maximum of 1.5 hours. For collected sweat patches from each region and time interval, total sweat contained inside each patch was measured. On the basis of the measured volume of sweat and the time interval, regional sweat rate was computed. Collected sweat from each region and time interval were further analyzed by ICP-MS to measure concentrations of Na+ and K+.

For iontophoresis sweat analysis, regional sweat was collected for a total of 28 to 32 min using our sweat patches. For time-dependent off-body analysis, subjects were wiped and cleaned with alcohol swap and DI water every 3 to 5 min, and new sweat patches were attached. Sweat from each timed sample was collected from each patch for further analysis by ICP-MS, LC, or glucose sensors.

For off-body sweat glucose measurement, 10 μl of collected sweat was injected onto the sensor surface, and the current response was recorded by benchtop electrochemical measurement instruments. Following measurement in sweat, a large volume of glucose stock solution (~1 ml) was introduced to overwhelm the glucose contribution from the small sweat volume. Small volumes of high-concentration glucose stock solution were successively injected to calibrate each sensor individually and then to back-calculate the glucose concentration indicated by the original current response to sweat.

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