The circadian clock is an internal timekeeping system that generates ~ 24-h cycles in physiology and behavior, maintaining a remarkably consistent period across physiological conditions and temperature1. Sodium chloride (NaCl) is a key physiological ion whose concentration varies across species, yet its influence on circadian rhythms remains poorly understood. Using a reconstituted cyanobacterial oscillator composed of KaiA, KaiB, and KaiC, we investigated how NaCl modulates circadian period and temperature compensation. Increasing NaCl concentrations progressively shortened the circadian period without substantially affecting oscillation amplitude, mimicking the effects of KaiB titration. While temperature compensation was maintained across varying KaiB concentrations, changes in NaCl partially disrupted temperature compensation, as Q₁₀ values correlated positively with salt concentration. We propose that NaCl perturbs the equilibrium of KaiB conformations and oligomerization, normally stabilized across physiological temperatures, thereby modulating circadian period and temperature compensation. These findings provide insight into how physiological salt levels influence circadian timing and drive the diversification of clock proteins across species.
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