The pulsation ratio of a milking machine affects milk flow and milking time, and has also been reported to influence teat condition and milk somatic cell count (SCC). However, most studies comparing pulsation ratios have been performed on conventional cluster milking (whole-udder level), where effects such as deteriorated teat end condition and increased milk SCC are likely to be caused by over-milking on teats that are emptied faster than the other teats. When the teat cups are detached from each udder quarter separately which can be done in automatic milking systems (AMS), the risk of over-milking, especially in front teats, may be significantly reduced. This study investigated the effects of pulsation ratio on teat end condition, milk SCC, milk yield, milking time and milk flow in an automatic milking system where each udder quarter is milked separately. In total, 356 cows on five commercial farms were included in a split-udder design experiment comparing three pulsation ratios (60:40, 70:30 and 75:25) with the standard pulsation ratio (65:35) during 6 weeks. Pulsation rate was 60 cycles/min and vacuum level 46 kPa. The 70:30 and 75:25 ratios increased peak and average milk flow and the machine-on time was shorter with 75:25, while both peak and average milk flows were lower and machine-on time was longer with the 60:40 ratio. No negative effects on teat condition or milk SCC were observed with any of the pulsation ratios applied during the study. Thus it is possible that increased pulsation ratio can be used to increase milking efficiency in AMS where quarter milking is applied.