Acoustic tweezers facilitate a noninvasive, contactless, and label-free method for the precise manipulation of micro objects, including biological cells. Although cells are exposed to mechanical and thermal stress, acoustic tweezers are usually considered as biocompatible. Here, we present a holistic experimental approach to reveal the correlation between acoustic fields, acoustophoretic motion and heating effects of particles induced by an acoustic tweezer setup. The system is based on surface acoustic waves and was characterized by applying laser Doppler vibrometry, astigmatism particle tracking velocimetry and luminescence lifetime imaging. In situ measurements with high spatial and temporal resolution reveal a three-dimensional particle patterning coinciding with the experimentally assisted numerical result of the acoustic radiation force distribution. In addition, a considerable and rapid heating up to 55 °C depending on specific parameters was observed. Although these temperatures may be harmful to living cells, counter-measures can be found as the time scales of patterning and heating are shown to be different.