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Hyperpolarized (HP) xenon-129 (¹²⁹Xe), when dissolved in blood, has two NMR resonances: one in red blood cells (RBC) and one in plasma. The impact of numerous blood components on these resonances, however, has not yet been investigated. This study evaluates the effects of elevated glucose levels on the chemical shift (CS) and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mi mathvariant="normal">T</mi><mn>2</mn><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> relaxation times of HP ¹²⁹Xe dissolved in sterile citrated sheep blood for the first time. HP ¹²⁹Xe was mixed with sheep blood samples premixed with a stock glucose solution using a liquid–gas exchange module. Magnetic resonance spectroscopy was performed on a 3T clinical MRI scanner using a custom-built quadrature dual-tuned ¹²⁹Xe/¹H coil. We observed an additional resonance for the RBCs (¹²⁹Xe-RBC1) for the increased glucose levels. The CS of ¹²⁹Xe-RBC1 and ¹²⁹Xe-plasma peaks did not change with glucose levels, while the CS of ¹²⁹Xe-RBC2 (original RBC resonance) increased linearly at a rate of 0.015 ± 0.002 ppm/mM with glucose level. ¹²⁹Xe-RBC1 <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mi mathvariant="normal">T</mi><mn>2</mn><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> values increased nonlinearly from 1.58 ± 0.24 ms to 2.67 ± 0.40 ms. As a result of the increased glucose levels in blood samples, the novel additional HP ¹²⁹Xe dissolved phase resonance was observed in blood and attributed to the ¹²⁹Xe bound to glycated hemoglobin (HbA_1c).