Mixing or regrouping of calves from different pens is a common animal management practice on the farm, which frequently occurs after weaning and has a negative effect on calve welfare. Social integration before regrouping may relieve stresses, but more evidences are needed to verify this hypothesis. The present study aimed to investigate acute physiological and behavioral variations of individually- or group-housed calves after being introduced into a mixed group. A total of 132 postnatal calves were randomly divided into groups of 1, 3, 6 and 12 animals (S, G3, G6, and G12; 6 replicates in each group) until 59 days of age. At 60 days of age, every two replicates from different groups (S, G3, G6 and G12) were introduced in a larger pen which containing 44 of the aboved experimental calves. Before and after regrouping, physiological parameters of stress, including heart rate (HR), saliva cortisol (S-CORT), saliva secretory immunoglobulin A (SIgA), interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) levels, and behavioral responses were recorded. After regrouping, HR and S-CORT increased immediately (P