Cement-based 3D printing (additive manufacturing) has drawn significant attention in recent years as an emerging construction technology because of its potential environmental and economic benefits over the traditional cast-in-place concrete construction. Most research to date has focused on the macroscale properties of the printed structure. However, the performance of the printed materials depends on the properties of the individual filaments. In this study, grid nanoindentation coupled with scanning electron microscopy and energy dispersive X-ray spectroscopy was used to determine the effects of the printing process through extrusion on the local elastic indentation modulus and hardness of filaments in 3D printed cement paste structures. Dynamic changes in the water-to-cement ratio during the extrusion process combined with stress-induced dissolution of the cement particles led to variation in the median modulus of the printed filaments, with filaments having values greater than 23 GPa or lower than 20 GPa. Extrusion through a small diameter nozzle affected the mesoscale assemblage of the primary hydrate phases and led to printed filaments having a more uniform mesoscale packing of calcium silicate hydrates (C–S–H) with the disappearance of loosely packed C–S–H compared to their traditionally cast counterparts.