OBJECTIVE We examined whether the endogenous circadian timing system modulates proxies of mood vulnerability and well-being. METHODS Nineteen healthy participants (mean age: 26.6 years [23.0-30.2], seven females, body-mass index: 22.8 kg/m2 [21.1-25]) completed a laboratory protocol with a 32-hour Constant Routine, a stringently controlled protocol designed to isolate assessment of endogenous circadian rhythms. We assessed hourly anxiety- and depression-like mood (i.e., those typically observed in depression and anxiety) and well-being (i.e., associated with mental fatigue and physical comfort). RESULTS Significant endogenous circadian rhythms were observed in anxiety-like and depression-like mood, as well as well-being (p values from the mixed-model analysis using false discovery rates < .001). Post-hoc comparisons revealed more anxiety-like and depression-like mood during the circadian phase 60°-75° (∼8-9 a.m.), and more mental fatigue and less physical comfort during the circadian phase 30°-60° (∼6-8 a.m.). CONCLUSIONS Our data indicate endogenous circadian rhythms in anxiety-like and depression-like mood and well-being in healthy young adults. Future studies will help establish circadian-based therapeutics for individuals experiencing mood and anxiety disorders.