Speciation of particulate phosphorus in soils and aquatic sediments by sequential extraction techniques is commonly used to address both fundamental and applied problems. It has been suggested, however, that sequential extraction techniques can produce misleading results due to the generation of new solid phases during the extraction process. In this study, a well-established fractionation technique introduced by Psenner et al. (1984) for crystalline sediments was reevaluated in four calcite-rich lake sediments using a set of additional extraction tests and structural analyses. Extended tests using a sediment artificially enriched with Al and Ca, regarded as model sediment, showed a distinct overestimation of Ca-bound P (HCl-TP) and concomitant underestimation of metal oxide-bound P (NaOH-SRP). The formation of new solid phases during extraction was evident by P addition to the alkaline extract, change of volume-to-solid ratio, and variation of extraction time. Examination of the model sediment with infrared and X-ray spectroscopy demonstrated that Ca-bound P was not present before the NaOH step. Apatitic structures were detected by IR spectroscopy in the model sediment after NaOH extraction. In contrast, in the three natural calcite-rich sediments with high HCl-TP portions, underestimations of NaOH-SRP were not detected with the standard protocol. Therefore, a modification of the Psenner method does not appear necessary for most conditions. In hardwater lakes, however, especially those restored with Al and Fe salts, a shift from NaOH-SRP to HCl-P cannot be excluded. Our study provides helpful tools to identify such artifacts and thus to prevent misinterpretation of fractionation results.