In any therapeutic setting, the outcome depends in part on the therapeutic alliance, characterized by mutual understanding, empathy and trust among the participants, which also manifests itself through physiological synchronization (PS) processes involving breathing, heart, and brain. This study examined the dynamics of heart rate variability (HRV) synchronization patterns during horse-assisted therapy. We explored the impact of horse preference of the therapist, and levels of early life adversity (ELA) on PS relationships across participants, riding therapists and therapy horses. Our sample of 43 participants engaged in standardized horse-assisted therapy sessions facilitated by three riding therapists and four therapy horses. PS was operationalized through cross-wavelet power analyses across the different dyads. The results showed that the riding therapist's preference for a particular therapy horse was associated with less HRV synchronization between this therapy horse and the respective riding therapist. Strong synchronization between horse and therapist was associated with strong synchronization between horse and participant and between therapist and participant. We found an association between ELA and HRV synchronization between participants and therapists, with HRV synchronization being lower in individuals with higher levels of ELA. This effect of ELA was not found for HRV synchronization between participants and horses. These findings contribute to a better understanding of the therapeutic mechanisms underlying horse-human interactions and have implications for optimizing therapeutic interventions in clinical settings.