Aims: To evaluate whether participant characteristics and way of expressing circulating fatty acids (FA) influence the strengths of associations between self-reported intake and circulating levels of linoleic acid (LA), alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Methods: Cross-sectional analyses were performed in pooled data from the CODAM (n = 469) and Hoorn (n = 702) studies. Circulating FA were measured by gas liquid chromatography and expressed as proportions (% of total FA) and concentrations (μg/mL). Dietary intakes were calculated from a validated food frequency questionnaire. Effects of participant characteristics on associations between dietary and circulating FA were calculated using interaction analyses. Results: Standardized regression coefficients between dietary FA and proportions of circulating FA (% of total FA) were LA β = 0.28, ALA β = 0.13, EPA β = 0.34, and DHA β = 0.45. Body mass index (BMI), waist circumference, and presence of CVD influenced associations for LA; gender influenced LA, EPA, and DHA; alcohol intake influenced LA and DHA; and glucose tolerance status influenced ALA (p values interaction <0.05). Coefficients for circulating FA as concentrations were LA β = 0.19, ALA β = 0.10, EPA β = 0.31, and DHA β = 0.41. Conclusions: This study suggests that characteristics such as BMI, alcohol intake, and expressing circulating FA as proportions or concentrations, influence associations between dietary and circulating FA.