This work focuses on the application of accumulation-based and trip-based macroscopic fundamental diagrams (MFD) approaches to real transportation networks and discusses the calibration of the MFD shape and trip lengths estimation using a thorough validation of the network dynamics with microsimulation data. This work not only investigates a classical unimodal approach to fit the production MFD, but also a bimodal MFD curve. Different methods of calibrating trip lengths in the reservoir are introduced to study the influence of trip lengths estimation on the accuracy of MFD models. MFD models are validated against microsimulations that are carried out using real origin–destination (OD) matrix and demand estimated from the data of Lyon city in France. The proposed bimodal production MFD curve captures the hysteresis in the production MFD to a good extent. Subsequently, it is shown that the refined description of trip lengths gives more accurate estimates of accumulation evolution for the trip-based approach. Finally, a case is presented with a modified OD matrix to study the effect of OD matrix changes on accuracy of MFD simulations.