Large comparative studies in animal ecology, physiology, and evolution often use animals reared in the laboratory for many generations; however, the relevance of these studies hinges on the assumption that laboratory populations are still representative for their wild living conspecifics. In the present study, we investigate if laboratory-maintained and freshly-collected animal populations are fundamentally different and if data from laboratory-maintained animals are valid to use in large comparative investigations of ecological and physiological patterns. Here, we obtained nine species of Drosophila with paired populations of laboratory-maintained and freshly-collected flies. These species, representing a range of ecotypes, were then assayed for four stress tolerance traits, two body size traits, and six life history traits. For all of these traits, we observed small differences in species-specific comparisons between field and laboratory populations; however, these differences were unsystematic and laboratory maintenance did not eclipse fundamental species characteristics. To investigate if laboratory maintenance influence the general patterns in comparative studies, we correlated stress tolerance and life history traits with environmental traits for the laboratory-maintained and freshly-collected populations. Based on this analysis we found that the comparative physiological and ecological trait correlations are largely similar irrespective of provenience. This finding is important for comparative biology in general because it validates comparative meta-analyses based on laboratory-maintained populations.