Animal welfare is a growing concern in aquaculture practices. Stress induced by handling or transportation can lead to negative impacts on the sector. Zebrafish has raised as an important aquaculture model, but still with few focus on its stress response in early life stages. Therefore, the objective of this study was to improve the evaluation of different markers of the stress response after a stress event in a zebrafish larvae model. Zebrafish larvae (96 hpf) were vortex-stimulated for 1 min at 200 rpm for acute stress induction. After 10 min, 1- and 4-h behavioral larvae outcomes and larvae were sampled to the following quantification: levels of cortisol, lactate, glucose and biochemical biomarkers (reactive oxygen species, superoxide dismutase, catalase, glutathione peroxidase, lipidic oxidation level and protein carbonylation, glutathione s-transferase, acetylcholinesterase, lactate dehydrogenase and ATPase), and the metabolic rate. The cortisol, glucose, and lactate levels had no alterations. At the behavioral level, an increase in the distance swam and in the speed was observed and the metabolic rate also increased according to the behavioral outcomes. The ATPase and GST activity showed a decrease in their activity, probably through osmoregulation changes related to the hypothetic adrenocorticotropic hormone downregulation. Overall, the acute vortex stimulation at low speed induced an early stress response independent of the HPI-cortisol pathway. In addition, this study shows zebrafish early life stages as a sensitive model to acute vortex stimulation, identifying altered parameters which can be used in future work to assess the effect on animal welfare in similar acute situations.