<p>Anxiety symptoms exhibit day-night variation, often peaking in the late afternoon or evening. Despite clinical recognition of anxiety’s diurnal variation, time of day is rarely considered in the design or interpretation of anxiety research. In this study, we used aged (10–12 month) Long-Evans male rats to examine the extent in which time of day, chronic (&gt; 15 weeks) high-fat diet feeding, and time-restricted feeding affect anxious-like behaviors in the elevated plus maze and the novelty box test. We find that anxious-like behaviors, such as closed-arm entries, are consistently higher during the animal’s inactive phase. This day-night variation was unaffected by high-fat diet feeding. Interestingly, restricting food to a 12-hour window did not invert the day-night variation in anxiety but reduced anxious-like behavior overall. These findings underscore the importance of multiple testing times when measuring anxiety and illustrate the relative resiliency of daily patterns in anxiety expression. Overall, we conclude that time of day modulates anxious-like behavior and should be considered in experimental designs and therapeutic interventions for anxiety.</p>