Daphnids are freshwater crustaceans used in toxicity tests. Although lethality and immobilisation are the most commonly used endpoints in those tests, more sensitive parameters are required for determination of sublethal acute effects of toxicants. The use of various physiological endpoints in daphnids is considered as a low-cost and simple alternative that meets the 3R’s rule (Replacement, Reduction, Refinement) criteria. However, currently there is no review-based evaluation of their applicability in toxicity testing. This paper presents the results on the most commonly determined physiological parameters of Daphnia in ecotoxicological studies and human drug testing, such as feeding activity, thoracic limb movement, heart rate, cardiac area, respiratory activity, compound eye, mandible movements and post-abdominal claw contractions. Furthermore, their applicability as promising endpoints in the assessment of water quality or drug testing is discussed.