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The 3Rs - Replacement, Reduction and Refinement - have become increasingly important in designing animal experiments. The Pavlov sling is thought to be a non-invasive method to restrain dogs for examinations. The aim of our study was to investigate whether laboratory Beagle dogs that had been trained to tolerate restraint by a Pavlov sling are stressed by this procedure and, furthermore, to analyze their behavior during this period. Five male and five female Beagle dogs were used, each three years of age. Animals were restrained in the Pavlov sling for 30 min on six days with an interval of at least two days. The following behaviors were recorded every minute for each session: postures of body, head, and ears, as well as state of eyes, tail, legs, and mouth. Additionally, the animals were observed for the occurrence of particular stress signs, including body shaking, sweating of the paws, increased saliva production, piloerection, blinking of eyes, snout licking, yawning, and panting. As an indicator for stress, salivary cortisol levels were measured before, during, and after each session. Our results show that for most behavioral parameters, e.g., body, leg, head, tail, and ear posture, the frequency of changes between different behavior patterns, as well as cortisol concentration, were not influenced by restraint in the Pavlov sling. Therefore, the Pavlov sling does not seem to be perceived as a stressful situation by the Beagle dogs. Our study demonstrates that under certain conditions the use of the Pavlov sling in trained dogs can substitute for more ordinary methods of immobilization, e.g., the use of narcotics.