[Objective] Serum ionized Ca (iCa) level measurements acquired using the ion‒selective electrode method should be obtained within 3 to 4 hours. In whole‒blood gas analysis, the influence of delaying iCa measurements for >3 hours was investigated. [Method] In 43 hemodialysis patients, 2 mL of whole blood was drawn from an internal shunt before dialysis using a balanced heparin‒coated syringe, and gas analysis was performed promptly. The sample was left for 3.5 hours at room temperature, and then gas analysis was performed again. [Results] Comparing the values obtained before and after the sample was left for 3.5 hours revealed the following changes: pH: from 7.375±0.034 to 7.304±0.035, HCO3: from 20.20±1.85 to 19.91±1.80, base excess: from -4.38±1.85 to -6.10±1.89, pCO2: from 35.39±3.72 to 40.81±4.12, and lactate from 1.32±0.34 to 3.02±0.44 (all p<0.001). Despite the presence of acidosis, the subjects’ iCa and pH‒corrected iCa levels decreased from 1.13±0.07 to 1.12±0.08 (p<0.03) and from 1.12±0.07 to 1.08±0.08 (p<0.001), respectively. [Conclusion] Delaying whole‒blood gas analysis for 3.5 hours resulted in increased lactate, decreased pH, and decreased pH‒corrected iCa levels.