This study evaluated the serum cortisol response to a single session of whole-body vibration (WBV) in healthy adult dogs. Ten healthy adult medium dogs, females and males, aged between 24 and 48 months and with body weight between 10.1 and 17.9 kg were used. A single WBV session at a frequency of 30 Hz for 5 min (3.10 mm peak displacement, 11.16 m/s2 peak acceleration, and 0.29 m/s velocity), then 50 Hz for 5 min (3.98 mm peak displacement, 39.75 m/s2 peak acceleration, and 0.62 m/s velocity), and finishing with 30 Hz for 5 min (3.10 mm peak displacement, 11.16 m/s2 peak acceleration, and 0.29 m/s velocity) was performed. Serum cortisol, heart and respiratory rate, and systolic blood pressure were evaluated at different time points: 1 min before WBV (1PRE) and 1 min (1POST), 60 min (60POST), and 360 min (360POST) after the WBV session. An increase (P = 0.0417) of the serum cortisol values was observed between 1PRE and 1POST and a decrease (P = 0.0417) between 1POST and 60POST and between 60POST and 360POST. However, the values remained within the reference range. The heart and respiratory rate and the systolic blood pressure remained unchanged. Our findings suggest that a single bout of WBV (5 min of 30 and 50 Hz) using a vibrating platform that delivered a vortex wave circulation does not modify the serum cortisol levels and clinical parameters of healthy adult dogs.