Epithelial tissue structure is the emergent outcome of the interactions between large numbers of individual cells. Experimental cell biology offers an important tool to unravel these complex interactions, but current methods of analysis tend to be limited to mean field approaches or representation by selected subsets of cells. This may result in bias towards cells that respond in a particular way and/or neglect local, context-specific cell responses. Here, an automated algorithm was applied to examine in detail the individual calcium transients evoked in genetically homogeneous, but asynchronous populations of cultured non-immortalized normal human urothelial cells when subjected to either the global application of an external agonist or a localized scratch wound. The recorded calcium transients were classified automatically according to a set of defined metrics and distinct sub-populations of cells that responded in qualitatively different ways were observed. The nature of this variability in the homogeneous cell population was apportioned to two sources: intrinsic variation in individual cell responses and extrinsic variability due to context-specific factors of the environment, such as spatial heterogeneity. Statistically significant variation in the features of the calcium transients evoked by scratch wounding according to proximity to the wound edge was identified. The manifestation of distinct sub-populations of cells is considered central to the coordination of population-level response resulting in wound closure.