Abstract During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a “double-hit” approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit–induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel–Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional “linearity” (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.