Disturbance of circadian rhythms by high-fat diets (HFD) promotes insulin resistance and β-cell deterioration. Time-restricted feeding (TRF), which confines food access to specific daily periods without reducing calories, may restore rhythmic metabolic function. However, the optimal feeding window for metabolic protection remains debated. This study evaluated whether evening TRF (eTRF)—feeding aligned with the rodents’ active phase—can re-entrain peripheral clocks, enhance metabolic stability, and preserve β-cell viability in C57BL/6 mice fed an HFD

Male C57BL/6J mice (n = 40) were randomly distributed into four groups: (1) control chow ad libitum (CON), (2) HFD ad libitum (HFD-AL), (3) HFD with morning TRF (mTRF; 07:00–19:00), and (4) HFD with evening TRF (eTRF; 19:00–07:00) for 16 weeks under 12:12 h light–dark conditions. Body weight, glucose and insulin tolerance, locomotor activity, and core temperature were tracked. Pancreatic histology assessed β-cell mass (insulin staining) and apoptosis (TUNEL). Islet transcriptomics analyzed circadian regulators (Bmal1, RevErbα) and endoplasmic stress genes (Ddit3, Atf4). Serum GLP-1, triglycerides, NEFA, and inflammatory cytokines (IL-6, MCP-1) were quantified. Statistical comparisons used one-way ANOVA with Bonferroni post-hoc tests.

Continuous HFD feeding induced obesity (+45%), fasting hyperglycemia (~170 mg/dL), circadian disruption, and ~30% β-cell loss. Morning TRF modestly improved glucose tolerance, whereas eTRF significantly normalized body-weight gain (+20% vs CON), fasting glucose (~115 mg/dL), and restored daily activity and temperature rhythms. eTRF reduced GTT area by 40% and enhanced insulin sensitivity by 35% compared to HFD-AL (p < 0.001). β-cell mass was maintained near control levels (95%), with normalized apoptosis rates. eTRF elevated Bmal1 and RevErbα (~1.8-fold) while suppressing Ddit3 and Atf4 expression. GLP-1 levels increased 30%, and serum lipids and cytokines declined 25–40%.

Evening-restricted feeding effectively re-establishes circadian synchrony, maintains β-cell mass, and improves glucose regulation in HFD-fed mice without caloric restriction. Aligning food intake with the active circadian phase represents a promising, non-pharmacological strategy for preventing metabolic syndrome and β-cell exhaustion.