PurposeStudies show that dairy fat consumed in the form of cheese reduce LDL-cholesterol concentration (LDL-c) compared to butter and mechanistic suggestions include the calcium content of cheese leading to enhanced faecal fat excretion. The aim of this study was to test the effect of varying the calcium content within a cheese, on faecal fat excretion as a primary outcome, and blood lipid markers, fasting glucose and calcium excretion as secondary outcomes.Methods7 healthy males (BMI 18–25) participated in this randomized, cross-over control intervention, of 3 × 2 week periods. Diets contained 240 g/day cheese; a High Calcium Cheese (HCC) diet, a Reduced Calcium Cheese (RCC) diet, and a control arm: Reduced Calcium Cheese + CaCO3Supplement (RCC + Supp) diet. Diets differed in calcium content and form but were otherwise controlled for energy and key macronutrients. Blood and 5-day faecal samples were collected.ResultsThere was no significant difference in faecal fat excretion (g/day) between the diets (P= 0.066). Percent fat of faecel excretion was higher after RCC + Supp (P= 0.016). None of the individual fatty acids were different. Fasting LDL-c was significantly lower following the HCC diet vs. the other arms (P= 0.002). Faecal Ca was different across all diets (P= 0.001), lowest after RCC, and greatest after RCC + Supp. No differences were observed for fasting blood parameters or changes in anthropometry.ConclusionVarying the calcium content within a cheese matrix significantly affected fasting LDL-c values. Results did not support higher faecal fat excretion as an underlying mechanism, but the high attrition rate was a limitation.Trial registererTrial Registered at ISRCTN.org, registration number ISRCTN11663659 on 12.07.2022. Retrospectively registered.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00394-023-03118-8.