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Hydrologic signatures are quantitative metrics that describe streamflow statistics and dynamics. Signatures have many applications, including assessing habitat suitability and hydrologic alteration, calibrating and evaluating hydrologic models, defining similarity between watersheds and investigating watershed processes. Increasingly, signatures are being used in large sample studies to guide flow management and modelling at continental scales. Using signatures in studies involving 1000s of watersheds brings new challenges as it becomes impractical to examine signature parameters and behaviour in each watershed. For example, we might wish to check that signatures describing flood event characteristics have correctly identified event periods, that signature values have not been biassed by data errors, or that human and natural influences on signature values have been correctly interpreted. In this commentary, we draw from our collective experience to present case studies where naïve application of signatures fails to correctly identify streamflow dynamics. These include unusual precipitation or flow regimes, data quality issues, and signature use in human-influenced watersheds. We conclude by providing guidance and recommendations on applying signatures in large sample studies.