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Wearable sweat sensors are essential for providing insight into human physiological health. The currently developed microfluidic sweat sensors have demonstrated the function of collecting and storing sweat. However, they detect more average concentrations of substances based on time periods, which leads to the fact that in situ real-time measurement for multiple biomarkers remains a grand challenge. Here, we propose a wearable epidermal microfluidic patch with integrated microfluidic pumps and micro-valves for accelerated and continuous collection of the sweat, where the micro-pumps ensure the complete separation of old and new sweat for real-time detection of real concentration of biomarkers in sweat. The biomarker concentration at different time periods is detected by introducing a burst valve, which is used to assist in the analysis of the real-time detection. A quantitative relationship between the minimum burst pressure difference required for sequential collection and the size of the microchannel structure is established to overcome the effects of additional resistance at the gas–liquid interface. Additionally, the sensing modules, including sodium ion, chlorine ion, glucose, and pH level in sweat, are integrated into the patch to realize in situ, real-time detection of multiple biomarkers in the human sweat, decoding the correlation between changes in substance concentrations and physiological conditions. This work provides a unique and simplifying strategy for developing wearable sweat sensors for potential applications in health monitoring and disease diagnostics. [ABSTRACT FROM AUTHOR]