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Accurate clinical differentiation between skew deviation and fourth nerve palsy (4NP) is critical in the acute and subacute settings.To determine the sensitivity and specificity of the upright-supine test to detect vertical misalignment changes using different head positions for the diagnosis of acute or subacute skew deviation vs 4NP.This multicenter study enrolled consecutive patients from Coimbra University Hospital Centre, Coimbra, Portugal, and Michigan State University, Lansing, within 2 months of presenting with vertical diplopia and diagnosed as having skew deviation or acquired unilateral 4NP. The study used nonmasked screening and diagnostic test results from June 1, 2013, to December 31, 2016. Data were analyzed from January 1, 2017, to June 30, 2017.A 50% or greater change in vertical misalignment between the upright and supine positions, with the head centered and tilted to either side. Measurements included the alternate prism and cover (APC) test, the double Maddox rod test, the APC test change index ([measurement upright − measurement supine] / [measurement upright + measurement supine]), and the APC test sensitivity and specificity.Of the 37 included patients, the mean (SD) age was 58 (14) years, and 26 (70%) were male. We enrolled 19 patients (51%) with skew deviation and 18 (49%) with 4NP. Eighteen patients with skew deviation (95%) showed additional ocular motor and/or neurological signs. When moving to the supine position, only 1 patient with skew deviation (5%) showed more than a 50% decrease of hypertropia with the head centered (APC test: sensitivity, 5%; specificity, 100%). Three patients with 4NP (17%) showed more than a 50% decrease of hypertropia with the head tilted toward the hypertropic eye, and 10 patients with 4NP (56%) showed more than a 50% increase of hypertropia with the head tilted toward the hypotropic eye. Change indexes were different between the skew deviation and 4NP groups for head tilt to the hypotropic eye (difference, −0.33 prism diopters; 95% CI, −0.43 to −0.20; P < .001). Cyclotorsion worsened in the supine position only in patients with skew deviation (hypertropic eye: difference, −7.6 prism diopters; 95% CI, −13.00 to −0.75; P = .01; hypotropic eye: difference, 8.2 prism diopters; 95% CI, 0 to 15.75; P = .03).The upright-supine test with the head centered is not a sensitive method to separate acute or subacute skew deviation from 4NP. Conversion of an incomitant vertical deviation in the upright position to a comitant vertical strabismus in the supine position in all head positions, as well as the absence of additional ocular motor and/or neurologic signs, may constitute a more useful clue.