Introduction: Accurate estimates of intra-individual variability are necessary for proper design of clinical trials and epidemiological studies where the stress biomarkers cortisol and dehydroepiandrosterone sulfate (DHEA-S) are measured for dyads of persons with dementia (PWDs) and their family caregivers (FCGs). The aim is to determine the number of consecutive sampling days required to detect effect differences in clinical trials, and to accurately estimate regression coefficients in epidemiological studies where stress biomarkers are exposure variables in regression models with future disease as outcome.Methods: Clinical trial data from dyads of PWDs and their FCGs were used. Salivary cortisol and DHEA-S samples were collected five days a week, for eight consecutive weeks. From this data, we created formulas and graphical tools for the number of required sampling days needed to detect effect differences, and we calculated number of days needed for regression coefficients to be estimated with < 10% bias.Results: A total of 5791 salivary samples from 34 dyads were used. For morning cortisol, five consecutive sampling days at baseline and an equal number of days at study termination is sufficient to detect a treatment difference > 5% of baseline level with > 20 dyads per group. When stress biomarkers are used in epidemiological studies at least six consecutive sampling days are required.Conclusion: Based on a large number of consecutive measurements of stress biomarkers we calculated the sufficient numbers of sampling days for clinical trials and for epidemiological studies to produce credible results. Our findings will aid researchers in the study design phase.