The morphologic and metabolic basis of the tumor microenvironment is multifaceted, and this complexity is magnified throughout tumor progression. Therefore longitudinal surveillance of this habitat during tumor growth will improve drug targeting. We used translational MRI methods in glioblastoma models to investigate time-dependent remodeling of the extracellular matrix and microvasculature in relation to interstitial acidification. With tumor progression, we detected extracellular volume reduction and vascular plasma flow increase, but extracellular pH and vascular permeability remained unchanged. These differential dynamic processes regulating morphologic and metabolic transformations of the tumor microenvironment suggest distinct time windows for mechanistic therapeutic targeting.