Non-uniform heat fluxes are commonly observed in thermo-electronic devices that require distinct thermal management strategies for effective heat dissipation and robust performance. The limited research available on non-uniform heat fluxes focus mostly on microchannel heat sinks while the fundamental component, i.e. a single microchannel, has received restricted attention. In this work, an experimental setup for the analysis of variable axial heat flux is used to study the heat transfer in a single microchannel with fully developed flow under the effect of different heat flux profiles. Initially a hot spot at different locations, with a uniform background heat flux, is studied at different Reynolds numbers while varying the maximum heat fluxes in order to compute the heat transfer in relation to its dependent variables. Measurements of temperature, pressure, and flow rates at a different locations and magnitudes of hot spot heat fluxes are presented, followed by a detailed analysis of heat transfer characteristics of a single microchannel under non-uniform heating. Results showed that upstream hotspots have lower tube temperatures compared to downstream ones with equal amounts of heat fluxes. This finding can be of importance in enhancing microchannel heat sinks effectiveness in reducing maximum wall temperatures for the same amount of heat released, by redistributing spatially fluxes in a descending profile.