The study was undertaken to investigate the effect of unequal and equal milking intervals on milk yield, composition, somatic cell count (SCC) and free fatty acid (FFA) level of milk in cows yielding 25 kg/day. Sixty-six spring-carved cows were assigned to 2 treatments for a 4-week period (April 16 to May 14). Cows on treatments 1 and 2 were milked at 16:8h and 12:12h between milking intervals, respectively. The average lactation yield was 5,037 kg. Both treatment groups grazed under similar conditions and were stocked at 4.49/ha. Individual cow milk yields were recorded on 5 days each week and gross chemical composition was measured on 2 successive morning and evening milk samples each week. SCC and FFA of individual cow milks was measured on 1 successive morning and evening milk sample each week. There was no difference between the 16:8h and 12:12h interval with respect to daily yields of milk, milk protein and lactose. Daily milk fat yield and concentration were reduced (P<0.05) by the 12:12h interval but protein, lactose and SCC were not affected. In conclusion, total daily milk production, protein and lactose contents, SCC and FFA level of milk from cows with an average daily yield of 25 kg were not affected by a 16:8h compared to a 12:12h interval. Milk fat content was reduced with the 12:12 h interval and this may offer a mechanism to manipulate milk fat concentration in the future.