BackgroundCluster analyses have become popular tools for data-driven classification in biological psychiatric research. However, these analyses are known to be sensitive to the chosen methods and/or modelling options, which may hamper generalizability and replicability of findings. To gain more insight into this problem, we used Specification-Curve Analysis (SCA) to investigate the influence of methodological variation on biomarker-based cluster-analysis results.MethodsProteomics data (31 biomarkers) were used from patients (n = 688) and healthy controls (n = 426) in the Netherlands Study of Depression and Anxiety. In SCAs, consistency of results was evaluated across 1200 k-means and hierarchical clustering analyses, each with a unique combination of the clustering algorithm, fit-index, and distance metric. Next, SCAs were run in simulated datasets with varying cluster numbers and noise/outlier levels to evaluate the effect of data properties on SCA outcomes.ResultsThe real data SCA showed no robust patterns of biological clustering in either the MDD or a combined MDD/healthy dataset. The simulation results showed that the correct number of clusters could be identified quite consistently across the 1200 model specifications, but that correct cluster identification became harder when the number of clusters and noise levels increased.ConclusionSCA can provide useful insights into the presence of clusters in biomarker data. However, SCA is likely to show inconsistent results in real-world biomarker datasets that are complex and contain considerable levels of noise. Here, the number and nature of the observed clusters may depend strongly on the chosen model-specification, precluding conclusions about the existence of biological clusters among psychiatric patients.