Ischemic preconditioning can exert a powerful protection against a subsequent period of ischemia, via repeated inflation and deflation of a blood pressure cuff. Most often, damages of ischemia-reperfusion injury and benefits of preconditioning are evaluated via endothelial function. The ambulatory blood pressure monitoring constitutes repeated bouts of ischemia for 24 h. We examined whether repeated bouts of ischemia accumulated over 24 h influenced endothelial function. Twenty-two apparently healthy non-medicated middle-aged subjects 41 (8) years participated in the study. This study was registered with ClinicalTrials.gov (NCT03303404). Flow-mediated dilation (FMD) was measured as an index of endothelium-dependent vasodilation. The ambulatory blood pressure monitoring device went through an average of 110 (13) inflation/deflation cycles, which resulted in 46 (6) min of cumulative ischemic stimuli. Following 24-h of ambulatory blood pressure monitoring, FMD did not change significantly 6.6 (2.0) vs. 6.8 (2.7)%. Similarly, shear rate and reactive hyperemia were unchanged. We concluded that ambulatory blood pressure monitoring did not influence endothelium-dependent vasodilation acting via ischemic preconditioning.