In this study, effects of weaning on behavioral and physiological stress parameters in young horses (foals) were determined. Foals were weaned either simultaneously without the presence of adult horses (group A, n = 6), or in the presence of two adult females familiar but unrelated to the foals (group B, n = 5), or weaned consecutively by removing two mother horses per day (group C, n = 6). Behavior, locomotion, salivary cortisol concentration, beat-to-beat (RR) interval, heart rate variability (HRV) and weight were determined. Group A foals lost weight for 2 days (mean ± SEM) - 8.3 ± 1.6 kg, p < 0.05. Weaning was followed by increased vocalization which was least pronounced in foals of group B (p < 0.05). Locomotion was most pronounced on weaning day in foals of group A and lowest in group B (p < 0.05). Weaning increased salivary cortisol concentration on the day of weaning in groups A and B and for 2 days in group C (p < 0.05). The RR interval decreased most pronouncedly in group A foals (p < 0.05). There were no consistent changes in HRV. Based on cortisol release and behavior, weaning is associated with stress but this was least pronounced in foals weaned in the presence of two familiar but unrelated adult female horses.