Syringe pumps are widely used biomedical equipment which offer low-cost solutions to drive and control flow through microfluidic chips. However, they have been shown to transmit mechanical oscillations resulting from their stepper motors, into the flow, perturbing device performance. In this work, unlike previous studies at lower flow rates, we have uncovered that the relative pressure fluctuation plateau from 5mL/h onwards to approximately 2% of the average pressure. Furthermore, we find that absolute pressure fluctuations increase as a non-linear monotonic function of kinematic viscosity at flow rates in the 5-25 mL/h range, while the relative pressure fluctuations peak at 1.25 cSt. Using a novel low-cost coded compressive rotating mirror (CCRM) camera, we investigated the effect of fluctuations in a hydrodynamic microfluidic separation device based on a cell-free layer concept. Using this high-speed imaging set-up, we quantified the cell-free zone width fluctuations at bifurcations. We demonstrated that these fluctuations have the same frequency and amplitude than the syringe pump induced pressure oscillations. Finally, to illustrate that pressure fluctuations degrade the separation efficiency in such devices, we demonstrate using milk samples, instances of particles diverted to undesired outlets. This work provides an insight into the effect of syringe pump fluctuations on microfluidic separation, which will inform the design of microfluidic systems and improve their resilience to pulsating or fluctuating flow conditions.