This study investigated the effects of Lenient Steam Injection (LSI) treatment at temperatures 70–150 °C on the enzymatic activities of the indigenous milk enzymes alkaline phosphatase, lactoperoxidase (LPO), xanthine oxidase (XO), lipoprotein lipase (LPL) and plasmin in comparison with two reference heat treatments of 63 °C for 30 s and of 72 °C for 15 s by indirect heating. Milk fat globule (MFG) size distributions and pH were also monitored. Alkaline phosphatase, LPO, XO and LPL activities decreased with increasing LSI temperature. Plasmin activity was increased at temperatures <80 °C and decreased at temperatures above 90 °C. Milk fat globule homogenisation was observed at temperatures above 110–130 °C.