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Caffeine is a substance present in several foods and drinks of common western diet. Although high caffeine concentrations induce anxiogenic properties in various species, the influence of the different baselines of anxiety levels on caffeine-mediated responses is poorly understood. The short-fin wild-type (WT) and leopard (leo) zebrafish populations present significant behavioral differences, in which leo shows exacerbated anxiety-like responses. Since behavioral neurophenotyping may be easily assessed in adult zebrafish by associating temporal and spatial three-dimensional reconstructions of locomotion, we investigated the effects of caffeine on exploration and anxiety-like behavior of WT and leo zebrafish. Moreover, the whole-body cortisol content was assessed in the absence and presence of caffeine. For this purpose, animals were acutely exposed to caffeine (25, 50, 100 and 200 mg/L) for 15 min and further tested in the novel tank. Endpoint data and 3D reconstruction plots revealed that caffeine was anxiogenic in both WT and leo populations by altering vertical swimming, freezing, and erratic movements depending on the concentration. Prominent anxiogenic effects during habituation to novelty were observed in WT, suggesting a fundamental role of the phenotype in caffeine-mediated neurobehavioral responses. Although untreated leo showed higher baseline cortisol levels than control WT, caffeine increased whole-body cortisol in both populations. Moreover, caffeine induced aberrant swimming profiles in WT and leo following 200 mg/L exposure, which could reflect nonspecific toxicity and/or seizure-like behaviors. Collectively, our novel findings show that caffeine effects in zebrafish differ in a population-dependent manner.Behavioral effects of caffeine were tested in WT and leo zebrafish populations.Behavioral neurophenotyping was analyzed by endpoints and 3D swimming traces.Caffeine increased anxiogenic-like behaviors and whole-body cortisol of zebrafish.Caffeine prominently impaired the habituation to novelty in WT.High caffeine concentrations induced abnormal behavior in zebrafish.