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The vitreous humor is a soft viscoelastic gel composed of a complex network of biomolecules (primarily collagen and polyhyaluronic acid, “hyaluronan”). While extensive progress has been made in identifying the components of the vitreous, lack of sufficient experimental methods has hampered previous efforts to quantitatively define its mechanical properties. To address this issue, we have developed a novel “cleat” tool geometry for dynamic shear rheometry. Using this geometry we find that the shear moduli of vitreous decline by roughly a factor of five to steady‐state values within an hour after removal from the eye. Steady‐state values (Porcine: G′ = 2.6 ± 0.9 Pa, and G″ = 0.65 ± 0.41 Pa, n = 9; Bovine: G′ = 6.5 ± 2.0 Pa, and G″ = 2.0 ± 0.6 Pa, n = 17) are significantly larger than previously reported. The decrease in modulus also correlates with a decrease in mass (65 ± 11 %, n = 8) that occurs spontaneously after the vitreous is extracted. Rheological findings, taken in combination with biochemical analyses suggest that hyaluronan, a ubiquitous polyelectrolyte in connective tissue, contributes to vitreous stiffness by inducing tension in the network in vivo.

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