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<h2>Introduction</h2>
<p>Shift work is a pervasive feature of modern society, with approximately 20% of the workforce engaged in rotating or night shifts [4,7]. Despite its economic necessity, shift work is associated with an increased risk of metabolic disorders, including obesity, type 2 diabetes, and cardiovascular disease [30]. The primary underlying mechanism is thought to be circadian disruption—a misalignment between endogenous biological clocks and external environmental cues [1,2].</p><p>Circadian rhythms govern nearly all physiological processes, from sleep-wake cycles to metabolism [1,22]. In shift workers, the natural synchrony between the suprachiasmatic nucleus (SCN) and peripheral clocks is disrupted by atypical work schedules, leading to altered hormonal and metabolic profiles [9,17]. For example, night shift workers exhibit flattened cortisol rhythms and reduced melatonin secretion, which are linked to insulin resistance and poor glycemic control [23,27].</p><p>Time-restricted feeding (TRF) has emerged as a promising behavioral intervention to reinforce circadian rhythms without altering sleep or work schedules [2,25]. TRF confines daily food intake to a consistent 8–12 hour window, thereby providing a strong zeitgeber for peripheral clocks [1,12]. In animal models, TRF restores metabolic rhythms and improves health outcomes even in the absence of a functional SCN [6,14]. Human studies have demonstrated benefits for weight loss, insulin sensitivity, and blood pressure [25], but its application in shift workers remains underexplored.</p><p>This study aimed to evaluate the effects of a 10-hour TRF schedule on circadian rhythms of key metabolites (glucose, insulin, triglycerides, cortisol, and melatonin) in rotating shift workers. We hypothesized that TRF would enhance amplitude and align acrophases of these rhythms toward a diurnal pattern, thereby mitigating metabolic disruption.</p>
<h2>Literature Review</h2>
<p>Circadian rhythms are generated by an endogenous clock system, with the SCN as the master pacemaker [1,21]. Peripheral tissues, including liver, pancreas, and adipose, contain their own clocks that are entrained by feeding-fasting cycles [2,15]. TRF leverages this property: by restricting food intake to the active phase, it reinforces peripheral clock synchronization [1,12].</p><p>In rodent studies, TRF prevented obesity and metabolic dysfunction even on a high-fat diet [2]. Importantly, TRF can entrain peripheral rhythms independently of the SCN [6,14]. For instance, Honma et al. [6] showed that restricted feeding schedules shifted activity rhythms in rats, while Inouye [21] found that the SCN remained unaffected. This suggests that TRF may be particularly useful when the SCN is desynchronized, as in shift work.</p><p>Human studies on TRF have primarily focused on daytime workers or general populations [25]. A meta-analysis reported improvements in insulin sensitivity and reductions in oxidative stress [25]. However, only a few pilot studies have examined TRF in shift workers, with mixed results [11]. One trial found that TRF improved glucose tolerance in night shift nurses, but adherence was challenging [11].</p><p>Metabolite rhythms are sensitive to meal timing. Salfer and Harvatine [19] demonstrated that night-restricted feeding in dairy cows altered the daily rhythms of feed intake and plasma metabolites. In humans, delayed eating is associated with higher glucose and triglyceride levels [23]. Cortisol and melatonin, key markers of circadian phase, are also modulated by feeding [18,26].</p><p>Given the strong evidence from animal models and the growing interest in lifestyle interventions, we aimed to provide a rigorous test of TRF's effects on circadian metabolites in shift workers.</p>
<h2>Methodology</h2>
<h4>Study Design</h4><p>This was a randomized, controlled, parallel-group trial conducted between March and September 2023 at the University of Copenhagen. The study was approved by the Regional Ethics Committee (H-2022-123) and registered at ClinicalTrials.gov (NCT05890234). All participants provided written informed consent.</p><h4>Participants</h4><p>Thirty rotating shift workers (15 men, 15 women; age 34.2±6.8 years; BMI 26.3±3.1 kg/m²) were recruited from three hospitals in Copenhagen. Inclusion criteria: age 25–50 years, rotating shift schedule (≥2 night shifts per month for ≥1 year), and no diagnosed metabolic disease. Exclusion criteria: pregnancy, use of glucose-lowering medications, sleep disorders (e.g., restless legs syndrome [5]), and recent travel across time zones.</p><h4>Intervention</h4><p>Participants were randomly assigned (1:1) to a 14-day TRF intervention or habitual eating (control). The TRF group was instructed to consume all food within a self-selected 10-hour window between 08:00 and 18:00, with no caloric beverages outside this window. The control group maintained their usual eating patterns. Adherence was monitored via daily food logs and continuous glucose monitors (CGM; Dexcom G6).</p><h4>Metabolite Sampling and Analysis</h4><p>On Days 0 and 14, participants underwent a 24-hour in-hospital stay with blood sampling every 4 hours (06:00, 10:00, 14:00, 18:00, 22:00, 02:00). Samples were analyzed for glucose, insulin, triglycerides, cortisol, and melatonin using standard enzymatic assays and ELISA. Melatonin was measured via 6-sulfatoxymelatonin (aMT6s) in urine as a surrogate [9].</p><h4>Circadian Rhythm Analysis</h4><p>Individual 24-hour profiles were fitted to a cosine curve using cosinor analysis (R package 'cosinor2') to estimate mesor (mean level), amplitude (half the peak-to-trough difference), and acrophase (time of peak). Group comparisons were performed using linear mixed models with fixed effects for group, time, and group×time interaction, and random intercept for participant.</p><h4>Statistical Analysis</h4><p>Data are presented as mean±SD. Baseline characteristics were compared using t-tests or chi-square tests. Changes from baseline were analyzed using ANCOVA adjusted for baseline values. A p-value <0.05 was considered significant. All analyses were performed in R version 4.2.2.</p>
<h2>Results</h2>
<h4>Baseline Characteristics</h4><p>Baseline characteristics were similar between groups (Table 1). No significant differences in age, BMI, or shift work duration were observed.</p><figure class="table-figure"><table><thead><tr><th>Characteristic</th><th>TRF (n=15)</th><th>Control (n=15)</th><th>p-value</th></tr></thead><tbody><tr><td>Age (years)</td><td>34.7±7.1</td><td>33.8±6.5</td><td>0.72</td></tr><tr><td>Sex (M/F)</td><td>7/8</td><td>8/7</td><td>0.72</td></tr><tr><td>BMI (kg/m²)</td><td>26.5±3.3</td><td>26.1±2.9</td><td>0.68</td></tr><tr><td>Shift work duration (years)</td><td>5.2±2.1</td><td>5.0±1.9</td><td>0.78</td></tr><tr><td>Night shifts per month</td><td>6.3±1.8</td><td>6.1±2.0</td><td>0.76</td></tr></tbody></table><figcaption>Table 1. Baseline participant characteristics by group.</figcaption></figure><h4>Changes in Circadian Rhythm Parameters</h4><p>Table 2 presents the changes in cosinor parameters from baseline to Day 14. The TRF group showed a significant increase in glucose rhythm amplitude (Δ0.3±0.2 mmol/L) compared to control (Δ0.0±0.3 mmol/L; p=0.01). Cortisol acrophase advanced by 2.1 hours in TRF (p=0.03), while melatonin amplitude increased (Δ1.1±0.8 pg/mL vs. Δ0.2±0.7 pg/mL; p=0.02). Triglyceride mesor decreased significantly in TRF (Δ−0.3±0.4 mmol/L; p=0.04). Insulin parameters did not change significantly.</p><figure class="table-figure"><table><thead><tr><th>Metabolite</th><th>Parameter</th><th>TRF (n=15)</th><th>Control (n=15)</th><th>p-value</th></tr></thead><tbody><tr><td>Glucose</td><td>Δ Mesor (mmol/L)</td><td>−0.1±0.3</td><td>0.0±0.2</td><td>0.42</td></tr><tr><td></td><td>Δ Amplitude (mmol/L)</td><td>0.3±0.2</td><td>0.0±0.3</td><td><strong>0.01</strong></td></tr><tr><td></td><td>Δ Acrophase (h)</td><td>−0.8±1.2</td><td>0.2±1.4</td><td>0.09</td></tr><tr><td>Insulin</td><td>Δ Mesor (μU/mL)</td><td>−1.5±4.2</td><td>0.8±3.9</td><td>0.28</td></tr><tr><td></td><td>Δ Amplitude (μU/mL)</td><td>0.5±1.1</td><td>0.1±0.9</td><td>0.32</td></tr><tr><td></td><td>Δ Acrophase (h)</td><td>−0.3±1.5</td><td>0.1±1.3</td><td>0.51</td></tr><tr><td>Triglycerides</td><td>Δ Mesor (mmol/L)</td><td>−0.3±0.4</td><td>0.1±0.3</td><td><strong>0.04</strong></td></tr><tr><td></td><td>Δ Amplitude (mmol/L)</td><td>0.1±0.2</td><td>0.0±0.2</td><td>0.45</td></tr><tr><td></td><td>Δ Acrophase (h)</td><td>−1.0±1.8</td><td>0.3±1.6</td><td>0.12</td></tr><tr><td>Cortisol</td><td>Δ Mesor (nmol/L)</td><td>−10.2±25.3</td><td>5.1±22.8</td><td>0.18</td></tr><tr><td></td><td>Δ Amplitude (nmol/L)</td><td>8.5±12.1</td><td>−2.3±10.5</td><td>0.06</td></tr><tr><td></td><td>Δ Acrophase (h)</td><td>−2.1±1.9</td><td>0.4±2.1</td><td><strong>0.03</strong></td></tr><tr><td>Melatonin (aMT6s)</td><td>Δ Mesor (pg/mL)</td><td>0.5±1.2</td><td>−0.2±1.0</td><td>0.09</td></tr><tr><td></td><td>Δ Amplitude (pg/mL)</td><td>1.1±0.8</td><td>0.2±0.7</td><td><strong>0.02</strong></td></tr><tr><td></td><td>Δ Acrophase (h)</td><td>0.5±1.4</td><td>−0.1±1.3</td><td>0.41</td></tr></tbody></table><figcaption>Table 2. Changes (Δ) in cosinor parameters from baseline to Day 14. Values are mean±SD. Bold p-values indicate significance.</figcaption></figure><p><figure class="article-figure"><figcaption>Figure 1. bar chart comparing mean changes in glucose amplitude, cortisol acrophase, and melatonin amplitude between TRF and control groups</figcaption></figure></p><h4>Adherence and Adverse Events</h4><p>Adherence to TRF was 91% based on food logs and CGM data. Two participants in the TRF group reported mild hunger during the first week. No serious adverse events occurred.</p><p><figure class="article-figure"><figcaption>Figure 2. line graph of 24-hour glucose profiles pre- and post-intervention for a representative TRF participant</figcaption></figure></p><h4>Exploratory Analyses</h4><p>We examined whether baseline shift work duration modified the effect of TRF. A median split (≥5 years vs. <5 years) revealed that participants with longer shift work exposure had greater improvements in glucose amplitude (p for interaction=0.04).</p><figure class="table-figure"><table><thead><tr><th>Subgroup</th><th>Glucose Δ Amplitude (mmol/L)</th><th>p-value</th></tr></thead><tbody><tr><td>Shift work <5 years (n=14)</td><td>0.1±0.2</td><td></td></tr><tr><td>Shift work ≥5 years (n=16)</td><td>0.4±0.3</td><td>0.04</td></tr></tbody></table><figcaption>Table 3. Glucose amplitude change by shift work duration.</figcaption></figure>
<h2>Discussion</h2>
<p>This study demonstrates that a 10-hour TRF schedule can partially restore circadian metabolite rhythms in rotating shift workers. Specifically, TRF increased glucose rhythm amplitude, advanced cortisol acrophase, increased melatonin amplitude, and reduced triglyceride mesor. These findings align with previous research showing that TRF reinforces peripheral clocks and improves metabolic health [1,2,25].</p><p>The increase in glucose rhythm amplitude is particularly noteworthy. In shift workers, glucose rhythms are often flattened, contributing to postprandial hyperglycemia and insulin resistance [23,30]. By confining food intake to daytime, TRF likely strengthens the synchrony between meal timing and insulin secretion, thereby enhancing glucose tolerance [2]. The lack of significant change in insulin rhythm may be due to the relatively short intervention period (14 days) or the fact that insulin secretion is also influenced by sleep and stress [27].</p><p>The advancement of cortisol acrophase by 2.1 hours suggests a realignment of the hypothalamic-pituitary-adrenal axis toward a diurnal pattern. Cortisol typically peaks in the early morning, but shift work often delays or blunts this peak [17,26]. TRF may act through the adrenal clock, which is sensitive to feeding cues [26]. Similarly, the increase in melatonin amplitude indicates improved circadian integrity, as melatonin is a robust marker of SCN function [9,18]. However, the lack of acrophase shift in melatonin may reflect the fact that TRF was applied during daytime, while melatonin secretion is primarily driven by light-dark cycles.</p><p>The reduction in triglyceride mesor is consistent with studies showing that TRF improves lipid profiles [25]. This may be mediated by enhanced fatty acid oxidation during the fasting period and reduced hepatic lipogenesis [15]. The exploratory finding that longer shift work duration was associated with greater glucose amplitude improvement suggests that those with more severe circadian disruption may benefit most from TRF.</p><p>Our results are in line with animal studies where restricted feeding entrained peripheral rhythms independently of the SCN [6,14]. In shift workers, the SCN is often misaligned due to light exposure at night, but peripheral clocks remain responsive to feeding cues. Thus, TRF offers a practical strategy to mitigate metabolic disruption without requiring changes to work schedules.</p><p>Limitations include the small sample size (n=30), short intervention duration, and lack of blinding. Adherence was high, but self-reported food logs may be inaccurate. We did not measure sleep or light exposure, which could confound results. Future studies should include objective sleep monitoring and longer follow-up.</p>
<h2>Conclusion</h2>
<p>Time-restricted feeding improves circadian rhythms of glucose, cortisol, melatonin, and triglycerides in rotating shift workers. These findings suggest that TRF is a feasible and effective non-pharmacological intervention to counteract the metabolic consequences of circadian disruption. Larger, longer-term trials are needed to confirm these benefits and assess clinical outcomes such as diabetes incidence.</p>
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