Fish screens are structures associated with pump stations and power plants, that prevent entrainment of fish, but may also be a source of physiological stress, if placed in locations of strong flow speeds that fish are unable to sustain swimming against over time. Herein, the acute response of Anguilla anguilla and Oncorhynchus mykiss to a 30-minute exposure to two water flow regimes was evaluated at the lowest level of the hypothalamus–pituitary–interrenal axis, from blood serum and skin mucus, in a controlled setup presenting a 45° vertically-angled fish screen. Cortisol response was species specific, regardless of the matrix employed. While the flow velocity factor did not describe any variance of eel data, and no statistically significant differences in cortisol concentrations were observed among eel groups, cortisol release in response to flume hydraulics followed a dose-dependent pattern in trout, with a large proportion of the variance described by the model. Mucus cortisol was highly and strongly correlated to serum levels of trout specimens subjected to the strongest flow. Given the established neuromodulatory and molecular roles of cortisol on major fitness-relevant processes, animal welfare implications may be severe, especially considering ever increasing exposure to chronic anthropogenic stressors, resulting in repeated and/or prolonged elevation of circulating glucocorticoids.