Background: Skin barrier function assessment is commonly done by measuring transepidermal water loss (TEWL). An important limitation of this method is the influence of intrinsic and extrinsic factors. Electrical impedance spectroscopy (EIS) is a lesser-established method for skin barrier function assessment. Some influential factors have been described, but no guidelines exist regarding the standardization of these measurements. Objective: To evaluate the effect size of daily routine activities on TEWL and EIS, as well as their correlation with age and anatomical differences. Methods: Healthy participants (n=31) were stratified into three age groups (18–29, 30–49, and ≥50 years). In a climate-controlled room, EIS and TEWL measurements were performed on the left and right volar forearm and abdomen. Results: Body cream application decreased TEWL and EIS values after 15 and 90 minutes. Skin washing decreased TEWL for 15 minutes and EIS values for at least 90 minutes. TEWL was increased 5 minutes after moderate to intense exercise. Coffee intake increased TEWL on the abdomen after 60 minutes. TEWL and EIS values did not correlate with participants’ age and no anatomical differences were observed. No correlation was observed between TEWL and EIS. Conclusion Body cream application and skin washing should be avoided at least 90 minutes prior to measurements of TEWL and EIS. Exercise and coffee intake should also be avoided prior to TEWL measurements. EIS may be a promising tool for skin barrier function assessment as it is less affected by daily routine activities than TEWL.