Horses are transported for many reasons, and loading habituation is potentially affecting animal welfare and human safety. Horses are neophobic but may be habituated and trained to perform complex behavioural tasks in novel environments. Before transporting, horses should preferably be habituated to the vehicle and transportation. However, not all horse owners know how this can be done or how to apply common ethological methodology. The aim of our study was to quantify loading problems experienced by horse owners through a survey, compare horse behaviour during the loading procedure at a veterinary clinic with at competition sites and to perform a controlled experiment to investigate the effects of a standardized loading habituation procedure. Part 1 of the study was a horse owner survey. In study 2 we observed horses loaded at competitions and horses loaded at a veterinary clinic to compare two populations with differing habituation levels. In part 3 of the study six 2–3 year-old Icelandic horses were observed during loading habituation and heart rate was measured during these procedures over three consecutive days. Swedish horse owners’ written survey answers (n = 99) showed that 21% experienced problems when loading their horses. Loading at the veterinary clinic took significantly longer (5.8 min) compared to at the competition site (28 s) (P < 0.0001). Horses showed a significantly higher number of evasive behaviours when being loaded at the clinic (16.0 SE ±9.4) compared to the competition site (1.3 SE ±0.5) (P < 0.0001). The Icelandic horses had significantly higher heart rate inside the trailer (73 bpm) compared to before loading (59 bpm) and when outside the trailer again (57 bpm) (P = 0.001). The time taken to load decreased significantly with number of times being loaded during the 3 experimental days (P = 0.001). We conclude that training, in accordance with learning theory, reduce fear when being loaded.