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The present study provides firstly a comprehensive review of studies on measuring the impacts of different biodiesel blends on exhaust emissions characteristics of urban busses under real-world operating conditions. Secondly, this paper discusses the errors that can be made in conducting case studies. Thirdly and finally, it shows lessons learned and provides guidelines to setup case studies, conduct the measurements, perform the statistical analysis and report the results to policy makers and the wider audience. To achieve climate change mitigation targets, using alternative fuels, e.g., biodiesel, hydrogen or electricty (EVs) for the urban fleets requires an in-depth analysis of the impacts under real-world operating conditions. Such experiments are generally very complex as numerous factors could directly or indirectly interfere with the results produced and potentially jeopardize the integrity of the research and the conclusions drawn. Results of the present research show that some vital parameters were ignored by many of the studies performed including the statistical uncertainties, driving cycle uncertainties and fuel uncertainties. Lack of appropriate experimental designs or clear assertions about the level of significance for differences in emissions/fuel consumption between alternative fuels (i.e. biodiesel) and the reference fuel used (i.e., diesel) could be regarded as the main weaknesses. Moreover, many other overarching and very influential factors (e.g., covariates/confounders) can interfere with the research outcomes as these were mostly overlooked by the reviewed studies. A careful and complete experimental design for assessments of alternative fueled vehicles (are critical when conducting real-world operating condition tests. The study findings help to formulate the guidelines for assessing real-world operating condition experiments to achieve the most feasibly and meaningful research outcomes that will have significant implication for local and global policy makers. The guidelines are of use for all types of research studies that want to evaluate the effects of alternative fuels for any transportation fleet.