The objective of this experiment was to investigate the effect of milking frequency and nutritional level on some aspects of animal health. Holstein-Friesian cows (n = 60) were assigned post calving to a factorial arrangement of treatments; twice a day (TAD) milking on a high or low nutritional level; once a day (OAD) milking on a high or low nutritional level. Milking characteristics were recorded daily. Blood samples to evaluate changes in the composition of the blood cells, milk leakage, udder tension and locomotory ability were measured on four occasions. Teat-ends were classified for hyperkeratosis (HK) monthly post partum. TAD had longer daily milking times (P < 0.001) compared with OAD cows. There was no effect of milking frequency or nutritional level on morning milking time, time to milk letdown or peak milk flow rate (P>0.05). High nutritional level cows had higher average flow-rates (P < 0.05) than low nutritional level cows. Neither milking frequency nor nutritional level affected HK (P>0.05). However, HK values were positively correlated with daily milking time for OAD cows for 6 months of lactation (P < 0.05). This correlation was significant (P < 0.01) for cows milked OAD on high nutrition during the peak lactation period. OAD cows had higher levels of milk leakage compared with TAD cows during the month of May (P < 0.01). Cows on high nutrition milked OAD showed higher udder firmness scores than cows milked once or twice daily at the low nutrition level in June and July (P < 0.05). OAD cows had higher locomotion scores compared with TAD cows (P < 0.001). Locomotion and udder firmness scores were significantly correlated for OAD in June (P < 0.05). OAD cows had lower blood lymphocyte counts, numerically higher counts of neutrophil and a higher monocyte count at peak lactation compared with TAD cows suggesting that OAD cows had altered immune responses. The increase in milk leakage, higher udder firmness and locomotion scores in conjunction with changes in blood cells, suggests that OAD milking may have caused some discomfort to the cows during peak lactation. A reduction in the concentrate input to OAD cows during this period could ameliorate this problem.