Abstract: This experiment investigated the effects of positive and negative tactile handling on the stress physiology and behaviour of dairy heifers. Forty-eight 5-14-month-old nonlactating Holstein-Friesian heifers were allocated to one of two handling treatments, either positive or negative tactile handling, over four time replicates. Handling was imposed twice daily, 2-5 min per session and involved moving animals individually along a 64 m outdoor route. The negatively handled heifers took longer to approach within 1 and 2 m of a stimulus person in a standard test, than their positively handled counterparts (P<0.001) and had a greater flight distance to an approaching stimulus (P<0.001). The time taken by the heifers to approach within 1 and 2 m of a familiar person was similar to that taken to approach within 1 and 2 m of an unfamiliar person in the standard test (P<0.05). There was a tendency for heifers to have a greater flight distance from the approaching unfamiliar person than from the approaching familiar person (P=0.06). The negatively handled heifers had greater (P<0.05) increases in total cortisol concentrations 5, 10 and 15 min after exposure to a human and had higher (P<0.05) free cortisol concentrations in the afternoon than the positively handled heifers. It is concluded that the nature of the human contact affects the subsequent behavioural response of heifers to humans. This behavioural response may extend to other humans through the process of stimulus generalisation, although there was some evidence of moderate discrimination. Negative handling results in an acute stress response in the presence of humans and also leads to a chronic stress response. Further research into the effect of these stress responses on milk production and welfare in fearful cows in a commercial situation is suggested.