Nutritional biomarkers are used for a variety of purposes in large-scale population surveys and epidemiologic studies as well as smaller clinical studies. The main reasons for using nutritional biomarkers are to provide measures of nutritional status that have less error than dietary data, nutrient status for nutrients with inadequate dietary data, to obtain a more proximal and integrated assessment of nutrient status that incorporates metabolism, to assess dietary change and compliance in intervention studies, and dietary intake for the validation of dietary questionnaires. However, often there is oversight by the investigators regarding biologic and laboratory issues, which have implications for the utility of nutritional biomarkers. This article reviews some of the physiologic issues that contribute to between-person variability in nutrient status and the utility and meaning of specimens from various body compartments. Issues related to the collection and storage of biologic specimens are addressed, although it is recommended that investigators contact laboratory colleagues at the beginning of any study for updated information. The necessity for blind quality surveillance of laboratory analyses beyond the normal procedures employed by collaborating laboratories also is addressed. The advantages and disadvantages of nutritional biomarkers are reviewed, especially in comparison with using dietary methodology.