Transformation of biomarkers (or their stability) during sewer transport is an important issue for wastewater-based epidemiology (WBE). Most studies so far have been conducted in the laboratory, which usually employed unrealistic conditions. In the present study, we utilized a pilot sewer system including a gravity pipe and a rising main pipe to investigate the fate of 24 pharmaceutical biomarkers. A programmable logic controller was used to control and monitor the system including sewer operational conditions and wastewater properties. Sequential samples were collected that can represent hydraulic retention time (HRT) of up to 8 h in a rising main and 4 h in a gravity sewer. Wastewater parameters and biomarker concentrations were analysed to evaluate the stability and transformation kinetics. The wastewater parameters of the pilot system were close to the conditions of real sewers. The findings of biomarker transformation were also close to real sewer data with seventeen biomarkers reported as stable while buprenorphine, caffeine, ethyl-sulfate, methadone, paracetamol, paraxanthine and salicylic acid degraded to variable extents. Both zero-order and first-order kinetics were used to model the degradation of unstable biomarkers and interestingly the goodness of fit R2 for the zero-order model was higher than the first-order model for all unstable biomarkers in the rising main. The pilot sewer system simulates more realistic conditions than benchtop laboratory setups and may provide a more accurate approach for assessing the in-sewer transformation kinetics and stability of biomarkers.