The mixed layer, or epilimnion, is a physical concept referring to an isothermal layer at the surface of a water body. This concept is ubiquitous within limnology, is fundamental to our understanding of chemical and ecological processes, and is an important metric for water body monitoring, assessment and management. Despite its importance as a metric, many different approaches to approximating mixed depth currently exist. Using data from field campaigns in a small meso-eutrophic lake in the UK in 2016 and 2017 we tested whether different definitions of mixed depth resulted in comparable estimates and whether variables other than temperature could be assumed to be mixed within the layer. Different methods resulted in very different estimates for the mixed depth and ecologically important variables were not necessarily homogenously spread through the epilimnion. Furthermore, calculation of simple ecologically relevant metrics based on mixed depth showed that these metrics were highly dependent on the definition of mixed depth used. The results demonstrate that an idealised concept of a well-defined fully mixed layer is not necessarily appropriate. The widespread use of multiple definitions for mixed depth impairs the comparability of different studies while associated uncertainty over the most appropriate definition limits the confirmability of studies utilising the mixed depths.