Abstract There is a growing interest in the neuroscience community on the advantages of multilayer functional brain networks. Researchers usually treated different frequencies separately at distinct functional brain networks. However, there is strong evidence that these networks share complementary information while their interdependencies could reveal novel findings. For this purpose, neuroscientists adopt multilayer networks, which can be described mathematically as an extension of trivial single-layer networks. Multilayer networks have become popular in neuroscience due to their advantage to integrate different sources of information. Here, we will focus on the multi-frequency multilayer functional connectivity analysis on resting-state fMRI recordings. However, constructing a multilayer network depends on selecting multiple pre-processing steps that can affect the final network topology. Here, I analyzed the fMRI dataset from a single human performing scanning over a period of 18 months (84 scans in total), and the second dataset of 25 subjects with 3 repeat scans. I focused on assessing the reproducibility of multi-frequency multilayer topologies exploring the effect of two filtering methods for extracting frequencies from BOLD activity, three connectivity estimators, with or without a topological filtering scheme, and two spatial scales. Finally, I untangled specific combinations of researchers’ choices that yield consistently brain networks with repeatable topologies, giving us the chance to recommend best practices over consistent topologies.

Assessing The Repeatability of Multi-Frequency Multi-Layer Brain Network Topologies Across Alternative Researcher’s Choice Paths