Hydrodynamic alignment of proteinaceous or polymeric nanofibrillar building blocks can be utilized for subsequent assembly into intricate three-dimensional macrostructures. The non-equilibrium structure of flowing nanofibrils relies on a complex balance between the imposed flow-field, colloidal interactions and Brownian motion. The understanding of the impact of non-equilibrium dynamics is not only weak, but is also required for structural control. Investigation of underlying dynamics imposed by the flow requires in situ dynamic characterization and is limited by the time-resolution of existing characterization methods, specifically on the nanoscale. Here, we present and demonstrate a flow-stop technique, using polarized optical microscopy (POM) to quantify the anisotropic orientation and diffusivity of nanofibrils in shear and extensional flows. Microscopy results are combined with small-angle X-ray scattering (SAXS) measurements to estimate the orientation of nanofibrils in motion and simultaneous structural changes in a loose network. Diffusivity of polydisperse systems is observed to act on multiple timescales, which is interpreted as an effect of apparent fibril lengths that also include nanoscale entanglements. The origin of the fastest diffusivity is correlated to the strength of velocity gradients, independent of type of deformation (shear or extension). Fibrils in extensional flow results in highly anisotropic systems enhancing interfibrillar contacts, which is evident through a slowing down of diffusive timescales. Our results strongly emphasize the need for careful design of fluidic microsystems for assembling fibrillar building blocks into high-performance macrostructures relying on improved understanding of nanoscale physics.