Reduced nicotinamide adenine dinucleotide (NADH) plays a crucial role in many biochemical reactions in human metabolism. In this work, a flow-mediated skin fluorescence (FMSF)–post-occlusion reactive hyperaemia (PORH) system was developed for noninvasive and in vivo measurement of NADH fluorescence and its real-time dynamical changes in human skin tissue. The real-time dynamical changes of NADH fluorescence were analyzed with the changes of skin blood flow measured by laser speckle contrast imaging (LSCI) experiments simultaneously with FMSF-PORH measurements, which suggests that the dynamical changes of NADH fluorescence would be mainly correlated with the intrinsic changes of NADH level in the skin tissue. In addition, Monte Carlo simulations were applied to understand the impact of optical property changes on the dynamical changes of NADH fluorescence during the PORH process, which further supports that the dynamical changes of NADH fluorescence measured in our system would be intrinsic changes of NADH level in the skin tissue.