Electrospinning has become a well-established method for creating nanofibrous meshes for tissue-engineering applications. The incorporation of natural extracellular components, such as electrospun pure collagen nanofibers, has proven to be particularly challenging, as electrospun collagen nanofibers do not constitute native collagen fibers anymore. In this study, we show that this detrimental effect is not only limited to fluorinated solvents, as previously thought. Rat tail collagen was dissolved in acetic acid and ethanol and electrospun at various temperatures. Electrospun collagen mats were analyzed using circular dichroism, enzymatic digestion, SDS-PAGE, western blotting, and Raman spectroscopy and compared to heat-denaturated and electrospun collagen in HFIP. Our data suggest that even non-fluorinated electrospinning solvents, such as acid-based solvents, do not yield structurally intact fibers. Interestingly, neither epithelial cells nor fibroblasts displayed a different cellular response to electrospun collagen compared to collagen-coated polyurethane scaffolds in F-actin staining and metabolic analysis using fluorescent lifetime imaging. The disruption of the structural integrity of collagen might therefore be underestimated based on the cell-material interactions alone. These observations expose the larger than anticipated vulnerability of collagen in the electrospinning process. Based on these findings, the influence of the electrospinning process on the distinct biochemical properties of collagen should always be considered, when ECM-mimicking fibrous constructs are manufactured.