This study showed that temperature influences the rate of separation of fat from natural whole milk during application of ultrasonic standing waves. In this study, natural whole milk was sonicated at 600 kHz (583 W/L) or 1 MHz (311 W/L) with a starting bulk temperature of 5, 25, or 40 °C. Comparisons on separation efficiency were performed with and without sonication. Sonication using 1 MHz for 5 min at 25 °C was shown to be more effective for fat separation than the other conditions tested with and without ultrasound, resulting in a relative change from 3.5 ± 0.06% (w/v) fat initially, of −52.3 ± 2.3% (reduction to 1.6 ± 0.07% (w/v) fat) in the skimmed milk layer and 184.8 ± 33.2% (increase to 9.9 ± 1.0% (w/v) fat) in the top layer, at an average skimming rate of ∼5 g fat/min. A shift in the volume weighted mean diameter (D[4,3]) of the milk samples obtained from the top and bottom of between 8% and 10% relative to an initial sample D[4,3] value of 4.5 ± 0.06 μm was also achieved under these conditions. In general, faster fat separation was seen in natural milk when natural creaming occurred at room temperature and this separation trend was enhanced after the application of high frequency ultrasound.