The natural behaviour of horses in response to danger is to take flight, and consequently human handlers can be injured. Reducing the flight response and general reactivity of horses is therefore likely to reduce the incidence of injuries to handlers. In this experiment we investigated the effect of handling foals in the first 2 days after birth on their subsequent response to handling, humans and novelty, and the foal-mare relationship. Standardbred foals were assigned to one of two groups, handled (H) (N = 22, 12 colts, 10 fillies) and control (C) (N = 22, 11 colts, 11 fillies). Handling took place 3 times/day on days 1 and 2 after birth for 10 min/session. Individual foals were gently restrained and stroked all over their body using bare hands and then a plastic bag and each leg was lifted once. C foals received no handling. C and H foals did not differ in their reaction to freeze branding at a mean age of 14 days. The approach and leave behaviour of mare-foal pairs were observed at pasture during week 5 to evaluate their relationship. Mares of H foals were less active in keeping the pair together than mares of C foals (GLM: F1,33 = 6.81; P < 0.05). At 6 weeks of age all colts were introduced to an arena, together with their mare, and their reaction to a novel object and an unknown human were tested. Treatment did not affect heart rate of foals or in mares. C foals initiated more suckling bouts than H when no human was present (Wilcoxon: Z = 2.44, N = 22, P < 0.05) indicating that they responded differently to the novel arena than H foals. However, there was no difference between H and C foals in their exploratory behaviour in the arena. When a human was present in the arena, H foals had a shorter flight distance than C foals (Z = -1.98, N = 22, P < 0.05) and tended to move further away from the mare (Z = -1.80, N = 22, P = 0.07). Handling of foals in the first 2 days after birth appeared to affect the foal-mare relationship and alter their perception of humans at a later age but did not alter their response to novelty or to handling. The effects of early handling of foals on the foal-mare relationship require further investigation.