<p>Transportation can be a significant stressor to laboratory animals. This study evaluated the efficacy of a five-week kennel conditioning protocol in mitigating stress induced by transportation in adult minipigs. Thirteen animals were randomly assigned to the conditioned (CND) or control (CTR) group. Only the CND group underwent conditioning in the kennel that would be used for transport. After the conditioning period, both groups were subjected to simulated transport. Stress responses were assessed through salivary cortisol levels at baseline, during kennel permanence, and during simulated transport, while heart rate variability (HRV) was continuously monitored. There were no significant differences observed in salivary cortisol levels between groups. Overall, neither group showed clear physiological evidence of transport-related stress. At baseline, CTR minipigs displayed higher Sample Entropy (SampEn), a nonlinear parameter of autonomic complexity, than CND minipigs. This suggests positive emotional arousal due to training. During transport, CND minipigs exhibited a significant intragroup increase in SampEn compared to baseline, indicating differences in autonomic regulation. These results suggest that repeated handling and exposure to non-aversive experimental procedures may contribute to stress resilience, potentially reducing the detectable impact of specific transport-conditioning protocols. Our results underscore the complexity of interpreting physiological stress indicators and support the use of multimodal approaches combining complementary welfare measures. Further studies are necessary to determine the relative contributions of targeted conditioning and general husbandry practices in shaping transport responses in biomedical minipigs.</p>