Multiple methods have been proposed to estimate pathway activities from expression profiles, and yet, there is not enough information available about the performance of those methods. This makes selection of a suitable tool for pathway analysis difficult. Although methods based on simple gene lists have remained the most common approach, various methods that also consider pathway structure have emerged. To provide practical insight about the performance of both list-based and structure-based methods, we tested six different approaches to estimate pathway activities in two different case study settings of different characteristics. The first case study setting involved six renal cell cancer data sets, and the differences between expression profiles of case and control samples were relatively big. The second case study setting involved four type 1 diabetes data sets, and the profiles of case and control samples were more similar to each other. In general, there were marked differences in the outcomes of the different pathway tools even with the same input data. In the cancer studies, the results of a tested method were typically consistent across the different data sets, yet different between the methods. In the more challenging diabetes studies, almost all the tested methods detected as significant only few pathways if any.