The relationship of farmed animals with humans has important implications for animal welfare and productivity. To investigate the short- and long-term effect of gentle interactions (stroking, talking in a gentle voice) during different life stages on the fear of humans, we tested heifers that had or had not experienced gentle interactions as calves in a previous experiment on a large commercial farm. We investigated a) whether the reduction in calves’ avoidance distance caused by gentle interactions was still detectable one year later, b) whether a second treatment with gentle interactions would be effective in reducing the avoidance distance of heifers and c) whether there were cumulative effects of the two treatment phases. We provided a total of 42 min of voluntary gentle interactions to 45 of 79 heifers, resulting in a cross-over design with four groups that had been stroked as calves; or as heifers; or both as calves and heifers; or not at all. We measured the avoidance distance before, 1 day after and 5 weeks after the treatment phase. There was no significant difference between heifers stroked or not stroked as calves in the avoidance distance measured before the treatment. All heifers that were stroked had an avoidance distance of 0 cm towards the experimenter after the treatment, and the decrease in avoidance distance was significantly higher than in non-stroked heifers (p < 0.001). The effect was persistent for at least 5 weeks and extended to an experimenter who was blind to the treatment (p = 0.03). Most heifers accepted the treatment for most of the time and stretched their necks while being stroked, which is a behavioural indicator of pleasure. The experience of gentle interactions as calves did not have a long-term effect on their avoidance distance before the treatment. Regular positive interactions may be necessary to maintain a good relationship with humans. Heifers seemed to perceive the treatment as pleasurable and displayed a very strong and consistent reduction in avoidance distance, indicating reduced fear of humans and increased confidence. Gentle tactile and vocal interactions may be a suitable method to improve heifers’ relationship with humans and their quality of life.