Purpose <p>Collagen fibrils provide mechanical integrity to the extracellular matrix in a variety of biological tissues. In biomedical research, quantification of fibril diameters is essential to describe remodeling of the matrix that can occur during development, disease, and healing. However, current statistical methods to analyze differences in the distribution of fibril diameters have significant limitations. This study evaluated a rigorous alternative method, Statistical nonParametric Mapping (SnPM), to compare fibril diameter distributions.</p> Methods <p>Randomly generated simulated datasets and experimental datasets of fibril diameter distributions were analyzed using both conventional tests and SnPM. Results from each method were compared.</p> Results <p>Conventional statistical tests to compare fibril diameter distributions demonstrated limitations. Comparison of average diameters detected overall shifts in distributions but not more nuanced changes, while analysis of binned relative frequencies showed dependency on the choice of bin width and location. In contrast, comparison of kernel density estimated probability density functions using SnPM both detected differences between groups and located those differences to specific ranges of fibril diameters.</p> Conclusion <p>Comparative analysis of groups of fibril diameter distributions using SnPM overcomes the limitations of current techniques and provides a reliable and rigorous technique for these data. Other potential applications for SnPM in biomedical research abound, extending to other distributional or multidimensional data.</p>