This study aimed to use a range of non-invasive monitoring technologies to investigate the behavioural and physiological responses of individually housed dairy calves to age at change in milk replacer (MR) feeding frequency. Forty-eight Holstein Friesian calves were individually penned and fed MR (625 g/d) as solids in one of three feeding regimes: (i) once-a-day feeding commencing at age 14 d (OAD14), (ii) once-a-day feeding commencing at age 28 d (OAD28) and (iii) twice-a-day feeding (TAD). Several behavioural (automatic activity sensors), physiological (infrared [IR] thermography and heart rate variability [HRV]) and haematological indicators were used to examine calf responses. Reduction in milk feeding frequency at 14 or 28 d of age increased daily concentrate intakes and drinking water consumption throughout the pre-wean period. Calf lying behaviour was unaffected by reduction in milk feeding frequency; however, TAD calves recorded a significant decrease in total daily lying time during the post-wean period compared with OAD28s. There was no effect of treatment on IR eye or rectal temperature throughout the experiment; however, there was an effect of age, with IR temperature decreasing as calf age increased. OAD14 calves tended to have decreased HRV at days 14 and 16, which is suggestive of an increased stress load. The findings suggest that under high levels of animal husbandry and whilst maintaining the same amount of milk powder/d (625 g/d), reduction in milk feeding frequency from twice to once daily at 28 d can occur without significant impact to behavioural, performance and physiological parameters assessed here.