There has been increasing pressure from the public against animal experimentation for testing and research purposes. The Three Rs (replacement, reduction, and refinement) principle is thought to be a key foundation concept in optimising the welfare of animals used in experiments. This retrospective study attempts to investigate the transition of the Three Rs in biomedical research through a review of articles published in Nature Medicine. We categorised all of the articles published in Nature Medicine from 1998 to 2003, on the basis of the pain and distress of the animals used in the experiments featured in the analysed article. We found there were no large fluctuations in the distribution of these categories over this time period. We also examined each article for the presence of a statement relating to the humane use of laboratory animals, and found that the number of articles which included such a statement dramatically increased in 2002. Over the years studied, there was a decreasing trend in the total number of animal types used for the experiments in the articles. Our results suggest that: a) more encouragement by journal editors might improve the attitude of scientists in terms of animal welfare; and b) the progress of replacement appears to be a more long-term effort in the field of biomedical research.