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Dietary deficiencies in ω-3 polyunsaturated fatty acids are known to effect retinal function including retinal ganglion cell (RGC) activity, which may have implications for glaucoma. In this study we consider retinal function after dietary manipulation and intraocular pressure (IOP) stress designed to compromise RGCs.Sprague-Dawley dams were fed either ω-3 sufficient (ω-3+, n=15) or deficient (ω-3−, n=16) diets 5 weeks before conception with pups subsequently weaned onto their mothers diets. At 20 weeks of age, acute IOP elevation was induced repeatedly through anterior chamber cannulation to 70 mm Hg for 1 hour on 3 separate occasions separated by 1 week. Electroretinograms were recorded 1 week after each IOP elevation to assay the photoreceptors (PIII), ON-bipolar cells (PII), and ganglion/amacrine cells (STR).Repeat IOP insult results in a specific RGC dysfunction (pSTR −14.5%, P<0.035) as does ω-3 deficiency (−26.4%, P<0.01). However, the combination of both causes an even larger RGC functional loss (−40.1%, P<0.001) than does either diet or IOP insult in isolation (P<0.001).Both ω-3 deficiency and repeat acute IOP insult cause RGC dysfunction and the combination of these factors results in a cumulative effect. Our data indicate that sufficient dietary ω-3 improves RGC function making it less susceptible to IOP insult.