The purpose of this randomized, double-blind, placebo-controlled trial was to evaluate the efficacy of a diet containing caseinate hydrolysate (CH) on signs of stress in 2 groups of dogs (defined as Anxious and Nonanxious), using physiological (serum cortisol and lysozyme, N:L ratios and heart rate) and behavioral parameters.From an initial group of 40 female Beagle dogs, ranging in age from 10 months to 4 years (mean 5 1.47 years; SD 5 0.53) belonging to a dog colony, 32 were selected for this study according to their level of anxiety. A group of 16 Anxious dogs and a group of 16 Nonanxious dogs were identified.A baseline period, aimed to obtain reference values of investigated parameters, preceded the experimental phase. Both groups (Anxious and Nonanxious) were divided into a treatment group, which received the diet containing CH, and a control group which received a placebo diet (PD). Anxious CH and PD groups were balanced for anxiety level. Each dog was evaluated 3 times a day at 4 weeks intervals (T1-T2-T3). Each evaluation lasted 2 days and involved a Reactivity Evaluation Form, a blood sampling, heart rate recording, and a 10-minute behavioral video recording. Results from Reactivity Evaluation Form scores showed that although at T1 Anxious dogs had significantly higher scores (Mann-Whitney test, P , 0.001) compared with Nonanxious dogs, no difference was found between Anxious dogs fed with CH diet and Nonanxious fed with PD or CH diet at T3. Behavioral observations evidenced some signs of improvement in Anxious dog fed with CH diet. Cortisol level significantly decreased in Anxious dogs fed with CH diet (Friedman test, P , 0.05). Individual differences in physiological measures of stress responses may have contributed to the large variability, making interpretation of these measures difficult. These results suggest that CH may be used as a functional ingredient alleviating stress in dogs.