The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes. Global collaborative studies offer a unique solution to this problem. Here, we present a global collaboration using the zebrafish (Danio rerio) novel tank test, a popular behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show that female zebrafish exhibit more anxiety-like behavior than males, highlighting sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, also influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (for example, rotifers) mitigated anxiety-like behaviors. Environmental stressors, such as noise and transportation, significantly impacted behavior. We recommend standardizing testing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds and minimizing stressors to improve the reliability of zebrafish behavioral studies.