Abstract Disclosure: W.W. Parksook: None. J.M. Brown: Consulting Fee; Self; Bayer, Inc. J. Milks: None. A. Moore: None. Y. Niebuhr: None. B. Honzel: None. A. Vaidya: Consulting Fee; Self; HRA Pharmaceuticals, Mineralys. Background: Calcium ion signaling is a critical factor required for the physiologic production of aldosterone. Emerging evidence has revealed that the majority of cases of primary aldosteronism involve pathogenic somatic mutations that ultimately increase calcium signaling and autonomous aldosterone production. Therefore, testing for primary aldosteronism that modulates calcium homeostasis may influence the diagnostic interpretation of the results. Method: Overweight to obese participants with metabolic risk factors and pre-hypertension or stage 1 hypertension (n=56) underwent a standard seated saline suppression test (SSST). Measurement of blood pressure, plasma aldosterone, serum calcium, as well as other factors, was performed at times 0, 120, and 240 minutes during SSST. Linear regression models were used to analyze the association between changes in calcium and each parameter. Results: Following 2 liters of saline infusion during SSST, all 56 participants (100%) had a significant decrease in serum calcium levels from baseline, with 26 (47%) developing new-onset frank hypocalcemia (calcium <8.6 mg/dL). The mean baseline serum calcium was 9.2 ± 0.3 mg/dL, which declined to 8.9 ± 0.4 mg/dL at 120 mins (P<0.001), and to 8.6 ± 0.4 mg/dl (P<0.001) at 240 mins when the SSST concluded. Consistent with the known vasopressor effect of calcium, greater drops in serum calcium levels were associated with lower blood pressure at 240 minutes (β;= 13.5, P<0.01 for DBP; β;= 10.7, P= 0.06 for SBP). Among those with post-SSST aldosterone levels <10 ng/dl (a categorical threshold used to exclude primary aldosteronism), greater reductions in serum calcium levels were associated with larger drops in aldosterone levels (β;= 9.9, P<0.05). Conclusion: The standard SSST induced a substantial decline in serum calcium levels in 100% of participants, with nearly half of all participants developing frank hypocalcemia at the end of saline infusion. Since calcium is known to play a critical role in physiologic and pathophysiologic production of aldosterone, these findings raise the question whether hypocalcemia induced by the acute loading of 2 liters of saline may confound the interpretation of aldosterone changes. Further research should focus on better understanding the diagnostic implications of these results and potential limitations of the SSST to exclude primary aldosteronism. Presentation: Friday, June 16, 2023