Microfluidic glucose biosensors and potassium ion selective electrodes were used in an in vivo study to measure the neurochemical effects of spreading depolarizations (SD), which have been shown to be detrimental to the injured human brain. A microdialysis probe implanted in the cortex of rats was connected to a microfluidic PDMS chip containing the sensors. The dialysate was also analyzed using our gold standard, rapid sampling microdialysis (rsMD). The glucose biosensor performance was validated against rsMD with excellent results. The glucose biosensors successfully monitored concentration changes, in response to SD wave induction, in the range of 10-400 μM with a second time-resolution. The data show that during a SD wave, there is a time delay of 62 ± 24.8 s (n = 4) between the onset of the increase in potassium and the decrease in glucose. This delay can be for the first time demonstrated, thanks to the high-temporal resolution of the microfluidic sensors sampling from a single tissue site (the microdialysis probe), and it indicates that the decrease in glucose is due to the high demand of energy required for repolarization.