Milking-related release of oxytocin, prolactin, and cortisol was studied following three pre-milking treatments. Six Murrah buffaloes were treated with direct application of milking cluster (O), a 1-min pre-stimulation (M), and combined feeding and pre-stimulation (MF). Machine milk yield, stripping yield and milk composition were recorded. Milk ejection occurred significantly earlier with MF than M and O (P<0·05; 2·50, 5·10 and 6·33 min, respectively). In all treatments, milk ejection occurred with small increases >3–5 ng/l in oxytocin concentration. Increase in oxytocin concentration over a threshold level and milk ejection occurred simultaneously and were closely correlated (r=0·83, P<0·05). There was a positive correlation between total time oxytocin concentration remained elevated over threshold levels and machine yield (r=0·86, P<0·05). For treatment O, milk ejection was inhibited during machine milking, while a marked increase in oxytocin occurred during hand stripping (6 and 16 ng/l, respectively). For treatment M, mean oxytocin concentrations remained unchanged during pre-stimulation but increased during subsequent machine milking and hand stripping (6·38, 18·06 and 12·36 ng/l, respectively). For treatment MF, although there was a 3·6-fold increase during pre-stimulation, oxytocin increased by 10-fold and 3-fold during machine milking and hand stripping, and was significant for machine milking (P<0·05, 17·32, 47·86, 18·13 ng/l, respectively). Milk-ejection-related cortisol release was visible only in treatment MF. For treatments O and M, prolactin concentration increased prior to the increase in oxytocin. The stripping yield was higher, and fat content in the stripping yield significantly lower, for treatment O indicating incomplete milking. Thus buffaloes are easily disturbed even by small changes in milking routines.