Suitable methods to realize a multi-dimensional fractionation of microparticles smaller than 10 μm diameter are still rare. In the present study, size and density fractionation is investigated for 3.55 μm and 9.87 μm particles in a sharp-corner serpentine microchannel of cross-sectional aspect ratio h∕w = 0.25 . Experimental results are obtained through Astigmatism Particle Tracking Velocimetry (APTV) measurements, from which three-dimensional particle distributions are reconstructed for Reynolds numbers between 100 and 150. The 3D reconstruction shows for the first time that equilibrium trajectories do not only develop over the channel width, i.e. in-plane equilibrium positions but also over the channel height at different out-of-plane positions. With increasing Reynolds number, 9.87 μm polystyrene (PS = 1.05 g cm−3) and melamine (MF = 1.51 g cm−3) particles focus on two trajectories near the channel bisector. In contrast to this, it is shown that 3.55 μm polystyrene particles develop four equilibrium trajectories at different in-plane and out-of-plane positions up to a critical Reynolds number. Beyond this critical Reynolds number, also these particles merge to two trajectories at different channel heights. While the rearrangement of 3.55 μm polystyrene particles just starts beyond Re > 140 , 9.87 μm polystyrene particles undergo this rearrangement already at Re = 100 . As the equilibrium trajectories of these two particle groups are located at similar out-of-plane positions, outlet geometries that aim to separate particles along the channel width turn out to be a good choice for size fractionation. Indeed, polystyrene particles of different size assume laterally well-separated equilibrium trajectories such that a size fractionation of nearly 100% at Re = 110 can be achieved.