This work shows that the longitudinal relaxation differences observed at very low magnetic fields between invasion/migration and proliferation processes on glioma mouse models in vivo are related to differences in the transmembrane water exchange basically linked to the aquaporin expression changes. Three glioma mouse models were used: Glio6 and Glio96 as invasion/migration models and U87 as cell proliferation model. In vivo proton longitudinal relaxation-rate constants (R1) at very low fields were measured by fast field cycling NMR (FFC-NMR). The tumor contribution to the observed proton relaxation rate, R1tum (U87: 12.26 ± 0.64 s−1; Glio6: 3.76 ± 0.88 s−1; Glio96: 6.90 ± 0.64 s−1 at 0.01 MHz), and the intracellular water lifetime, τin (U87: 826 ± 19 ms; Glio6: 516 ± 8 ms; Glio96: 596 ± 15 ms), were found to be good diagnostic hallmarks to distinguish invasion/migration from proliferation (p < 0.01 and 0.001). Overexpression of AQP4 and AQP1 were assessed in invasion/migration models, highlighting the pathophysiological role of these two aquaporins in water exchange that, in turn, determine the lower values in the observed R1 relaxation rate constant in glioma invasion/migration. Overall, our findings demonstrate that τin and R1 (measured at very low fields) are relevant biomarkers, discriminating invasion/migration from proliferation in vivo. These results highlight the use of FFC-NMR and FFC-imaging to assess the efficiency of drugs that could modulate aquaporin functions.