The aim of this study was to examine the relationships between flight distance (FD) and the reactions to direct contact with a human including the behavioural and physiological responses during handling in cows. Firstly, we measured FD of 16 Japanese Black cows and selected the 4 longest and 4 shortest FD cows (LFD and SFD, respectively). Secondly, a handling (gentle brushing) test was conducted 1 week after the FD test. Each cow was tethered during the handling test and brushed by same person who conducted the FD test. Behavioural responses (vocalization, tail swinging, head shaking and stepping) and heart rate (HR) of each cow were recorded before, during and after the handling periods (5 min, respectively). Behavioural responses and the mean HR of each period, the percentage of time where the HR during handling was higher than the mean HR before handling (PHR) and recovery times from the first peaks during handling to mean pre-handling HR (RT) for the LFD and SFD cows were compared by t-test. No difference was found for any behavioural response in each period between LFD and SFD cows. However, the mean HR of each period (before handling; 78.5 vs 61.3 bpm, P<0.05, during handling; 80.9 vs 59.0 bpm, P<0.01, after handling; 78.2 vs 59.7 bpm, P<0.05, LFD vs SFD, respectively) and PHR (LFD: 68.3 ± 40.9 %, SFD: 22.1 ± 9.6 %, P<0.05) were higher in LFD than in SFD cows. Furthermore, RT was longer in LFD (147.5 ± 133.6 s) than in SFD (16.3 ± 23.6 s, P=0.052). Consequently, flight distance was closely associated with the reaction to direct contact with a human and might reflect the psychological stress of cows.