Micro-computed tomography (µ-CT) is a non-destructive method to study 3D micro-structure of small samples, and has been used in a variety of fields including tissue engineering where the characterization of scaffolds hold great importance. The acquisition can be performed using different parameters such as different pixel sizes and rotation steps, and the obtained results may not be the same or even similar. In this study, using a wide range of acquisition parameters, tissue engineering scaffolds were characterized in a repeated-measures manner, and the effect of the acquisition parameters on the results of the analysis was shown in a systematic way. This study provides a critical assessment of the practice of the µ-CT analysis, and highlights that a fair comparison of results obtained by different acquisitions remains a challenge. Acknowledgements: This article is a result of the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). I. F. Cengiz thanks the Portuguese Foundation for Science and Technology (FCT) for the Ph.D. scholarship (SFRH/BD/99555/2014). J. M. Oliveira also thanks the FCT for the funds provided under the program Investigador FCT 2012 and 2015 (IF/00423/2012 and IF/01285/2015).