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ABSTRACT

The increased prevalence of a 24/7 society, monopolized by around-the-clock obligations, creates an environment that disrupts normal sleep patterns. The additional chronic exposure to artificial light via screen time has grown an epidemic of sleep deprivation. Only two-thirds of US adults regularly obtain adequate sleep (<7hrs) (10).  Over the last few decades, a plethora of studies have shown that acute and chronic sleep deprivation is a key contributor to multiple adverse health outcomes. These studies illustrate the effect of sleep deprivation on cardiometabolic and cardiovascular health across the lifespan (11,12). Adverse outcomes include higher body mass index, prevalence of obesity (9), development of insulin resistance (13), risk of type 2 diabetes mellitus (13), metabolic inflexibility (13), and elevated blood pressure (11,12).

Gabe Dennett (left) and Nate Goodwin (right). Photo credit: Jeremy Gasowski

While sleep deprivation has established markers for poor health outcomes, circadian misalignment has been far less explored. Circadian misalignment describes the mismatch between an individual’s internal circadian clock and external cues that aim to synchronize this clock that are disrupted by inconsistent sleep patterns (11). Few studies have isolated circadian misalignment independent of sleep deprivation to pinpoint its effects on autonomic function and metabolic flexibility, which sparked our interest in pursuing research on the topic with the support of a Summer Undergraduate Research Fellowship (SURF) in 2024.

One of the main reasons we chose to investigate irregular sleep patterns is that they are prevalent among the college population. Many college students follow a poor diet, consume alcohol, exhibit poor sleep patterns, and experience stress. Over 50% of college students in the US possess at least one cardiovascular disease risk factor, and the majority are oblivious to it or unconcerned about it (15). During the week, students might follow a rigorous sleep schedule because of class commitments, but on weekends, it is not maintained because they have no classes and/or because of social events. This irregularity of circadian patterns is termed social jet lag, which has been demonstrated to cause risk for cardiometabolic diseases. Therefore, it was our goal to design a study protocol that mirrors the common lifestyle of a young adult or college student who experiences relatively stable sleep duration (e.g., eight hours of sleep each night) but no standardization of sleep and wake times.

How Our Internal Clocks Shape Health and Performance

Recent investigations show that human physiology has evolved to function optimally around a circadian rhythm regulated by the light/dark cycle of the solar day, entrained by environmental cues termed zeitgebers—namely light, exercise, and feeding/fasting cycles (9,11). Light, being the most potent, is received from specialized cells in the eyes, containing melanopsin, a specialized pigment with high sensitivity to blue light waves (~460–480nm), which are emitted from our numerous device screens (11). When activated, these cells send light information to the suprachiasmatic nucleus, a portion of the brain that serves as the central circadian regulator. The suprachiasmatic nucleus then regulates peripheral clocks in virtually all organ systems and tissues with hormonal cascades involving melatonin and cortisol and with shifts in body temperature and energetics. This further adjusts peripheral clock timing in organs central to cardiometabolic health: the liver, pancreas, cardiovascular system, adipose tissue, and skeletal muscle (11). With proper rhythmicity and consistent introduction, this circadian system enhances the biological integrity and functional efficacy of the organism.

One of the most dominant circadian cues is the feeding/fasting cycle, which sets hormonal oscillations of cortisol, insulin, and glucagon (11). These hormones are central to glucose uptake and carbohydrate oxidation for energy metabolism (11). Sleep fluctuations interfere with such physiological processes, impairing the body’s ability to properly use fats and carbohydrates (1). When paired with increased sympathetic nervous system activation and inflammation, this may further set individuals on a path toward cardiometabolic diseases and may even impact exercise/athletic performance (3).

Inflammatory pathways and sympathetic nervous system dysregulation are two mechanisms related to elevated blood pressure that may be disrupted by circadian misalignment (6). Blood pressure is an important measure to consider regarding sleep health. Another important measure is heart rate variability (HRV), or the variation in time intervals between consecutive heartbeats, which is regulated by the balance of the parasympathetic and sympathetic nervous systems (5,14). These subdivisions of the autonomic nervous system, which are responsible for coordinating our involuntary physiological processes, operate like a seesaw; while one is more active, the other is suppressed. The sympathetic side is our “fight or flight” response, taking control in times of stress, heightened emotions, or physical demand, while the parasympathetic response slows our physiological processes toward “rest and digest” to focus on healing processes, energy conservation, and relaxation. During increased waking periods, the balance is shifted toward increased sympathetic nervous system activation, which is associated with cardiovascular disease risk (14). Markers of inflammation also are strong predictors of cardiovascular disease (8).

Clinically, inflammation can be detected through the presence of cytokines, small protein molecules crucial to our immune system, within the blood. C-reactive protein (CRP) is a pro-inflammatory cytokine found circulating in plasma and is associated with systemic inflammation (4). Increased CRP is associated with the development of high blood pressure and cardiovascular disease (4).

Previous circadian misalignment studies examining shift workers who work at night and sleep during the day found that CRP concentrations and blood pressure were augmented, but there was no change in heart rate variability (7,8). However, it remains unknown whether the typical fluctuation in sleep patterns in young adults influences the risks associated with cardiovascular health. Thus, a key aspect of our project was to determine if an acute exposure to circadian misalignment alters blood pressure, CRP, and heart rate variability in a college population. This was assessed by comparing biomarker variables to test the hypothesis that four days of misaligned sleep would increase blood pressure and CRP and reduce heart rate variability compared with circadian-aligned sleep. Another key aspect of our study was to determine if acute circadian misalignment influences exercise metabolism. This was assessed by implementing two separate sleep schedule protocols and comparing metabolic flexibility to test the hypothesis that four days of misaligned sleep would reduce fat oxidation compared with four days of circadian-aligned sleep.

Implementing an Effective Protocol

Figure 1. Participant characteristics

Thirteen recreationally active young adults aged eighteen to twenty-four completed our study, which was approved by the UNH Institutional Review Board. All were recruited via word of mouth, flyers, and social media. Participant demographics are displayed in Figure 1 and represent a healthy population. Individuals completed a health history questionnaire before beginning participation in the study. This self-reporting document screened for contraindications to data validation, as well as their safety in completing the study. We excluded those with any known cardiovascular disease, diabetes, neurological conditions, diagnosed insomnia, or sleep apnea, or who were currently taking sleep supplements or medications (e.g., melatonin, Ambien).

The study consisted of three visits to the Robert Kertzer Exercise Physiology Laboratory, in New Hampshire Hall on the �鶹app’s main campus over the course of roughly two weeks. There were two, four-day sleep phases (standardized sleep vs. misaligned sleep) separated by a washout of three to six days. A washout period describes a scheduled break in the protocol where participants resume habitual lifestyle activities to effectively “wash out” any effects of a previously employed sleep schedule.

The standardized sleep protocol required participants to go to sleep at their normal preferred bedtimes and achieve eight hours of sleep. The misaligned sleep protocol shifted ±1 hour onset/offset from the standardized sleep protocol times, while still maintaining eight hours of sleep. For example, if a participant chose their standardized sleep schedule as 10 p.m.–6 a.m., their misaligned sleep schedule would consist of 9 p.m.–5 a.m. and 11 p.m.–7 a.m. every other night for the four-night duration (Figure 2). The order in which participants completed the sleep schedules was randomized, and the washout period was determined based on participant and lab availability. On the last day of each phase, participants completed an ASA24 dietary recall. This is a self-reporting twenty-four-hour dietary guided recall technology developed by the National Cancer Institute where a participant reports any food, drink, and supplements consumed the prior day. We completed nightly check-ins via text message and phone calls to ensure participant compliance and comfort. 

Figure 2. Study design

On their first visit to our lab, we completed an informed-consent form with the participants, and they completed the health history questionnaire. Afterward, we measured participant characteristics such as height, weight, body composition, waist circumference, and a blood lipid panel. Then we took baseline measurements for our main variables to help familiarize the participants with the study protocol. The first consisted of resting heart rate variability and central blood pressure. A Polar H10 heart rate monitor was placed across the participant’s chest, and a brachial blood pressure cuff was placed on the left arm of each participant. Participants then lay in supine position in a dimly lit room for roughly fifteen minutes. The first five minutes consisted of quiet rest. This allowed every participant to be as relaxed as possible before we took the measurements, which ensured a controlled environment. After this, participants continued to rest quietly while we measured five minutes of spontaneous breathing HRV. Spontaneous breathing refers to normal, involuntary breathing that isn’t controlled by metronome or counting. Heart rate variability was obtained with the mobile application EliteHRV, which was connected to the aforementioned heart rate monitor via Bluetooth. After this, we took two central blood pressure measurements while the participant remained in quiet rest for another five minutes. Lastly, our mentor, Dr. Michael Brian, completed a blood draw to measure CRP levels.

After these measurements, we familiarized participants with the metabolic flexibility protocol. This test took place on a stationary bike attached to a metabolic cart to collect ventilation exchange. Participants wore a sealed mask that connects to the metabolic cart, allowing us to analyze the gas exchange between oxygen in and carbon dioxide out. This allowed us to calculate the amount of fat and carbohydrates they use, and measure exercise intensity from a metabolic level. This submaximal exercise test (Lode Excalibur Sport; Groningen, Netherlands) included a five-minute resting period, followed by five, five-minute stages of increasing exercise intensity, and was standardized across participants. Expiratory gas analysis measured relative oxygen consumption (VO₂R) and calculated substrate utilization (FatOx and CHOOx) during the metabolic flexibility protocol (Parvomedics True 2400; UT, USA). At the end of the baseline study visit, an ActivPal accelerometer (ActivPal; Glasgow, Scotland) was placed on the participant’s thigh to capture daily physical activity and sleep adherence. These thumb-sized accelerometers capture changes in movement, bodily posture, and activity. The ActivPals were also used to validate and quantify time-in-bed throughout each four-day sleep intervention and were worn for the entire duration of their participation. We concluded the visit by determining sleep schedules.

After each randomly ordered four-day sleep phase, participants returned to the lab, where we completed the same measurements of resting heart rate variability and central blood pressure, a blood draw, and the metabolic flexibility test. All data was stored and analyzed using Microsoft Excel. Heart rate variability data was imported to Kubios HRV software for analysis. Statistical analysis was completed using SPSS (IBM SPSS Statistics Version 27). We used a two-way repeated measures ANOVA test to compare fat oxidation during exercise between the two conditions. Paired t-tests compared heart rate variability, central blood pressure, and CRP between the two conditions. Two-way Repeated Measures ANOVAs compared oxygen consumption, fat and carbohydrate utilization, and heart rate from the metabolic flexibility test. Paired t-tests also compared resting variables between conditions.

Findings and Impact

We observed no difference in time-in-bed duration between the control and misalignment conditions, which indicates that participants were able to maintain sleep duration between interventions while shifting their alignment in accordance with the protocol. For heart rate variability, we analyzed two classifications of variables. The first classification, time domain, assesses the variability of the time duration between heartbeats. Not every heartbeat will occur within the same time frame as the previous one. Ideally, we want to see greater heart rate variability between heartbeats, because this reflects a healthy autonomic nervous system. We observed no significant differences in the time domain for our heart rate variability measures, implying that the misaligned condition induced little to no change.

Figure 3. Depicts high frequency (HF) and low frequency (LF) power between the aligned and misaligned sleep conditions.

The other classification is the frequency domain. The frequency domain can be defined as cyclic changes in heartbeats, which are measured in hertz and distributed into bands based on power. High-frequency power is a reflection of parasympathetic activity (rest and digest). Therefore, we were expecting to see a decrease in this variable. Low-frequency power is a reflection of mainly sympathetic activity (fight or flight). Therefore, we expected to see an increase in this variable. We observed a significant increase in low-frequency power and a significant decrease in high-frequency power during the misaligned sleep condition (Figure 3). These findings reflect a reduction in parasympathetic nervous system activity following acute circadian misalignment, which is associated with increased blood pressure and other detrimental cardiovascular outcomes. 

For our central blood pressure analysis, we observed no significant differences in systolic, diastolic, pulse, or mean pressures. These pressures are generic brachial blood pressure measures, as you would see at a doctor’s office. However, we did observe significant increases in augmentation pressure and augmentation index under the misaligned condition (Figure 4). Augmentation pressure refers to the amount that pressure increases because of reflected pressure waves. As blood leaves the heart, it is dispersed to peripheral tissue through smaller branches of blood vessels. This can be captured as a forward pressure wave, which will reflect off vessel branches, sending a pressure wave back toward the heart and, in turn, increasing pressure (much like waves in the ocean). This increase of pressure is referred to as augmentation, and the magnitude of the increase is influenced by pressure changes in peripheral vessels. 

Figure 4. Depicts central blood pressure variables between the aligned and misaligned sleep conditions. * indicates statistical significance between the two conditions (p<0.05).

Our central blood pressure data aligned well with our heart rate variability results, suggesting an increase in sympathetic nervous system activity. The increase in sympathetic nerve activity may be related to constriction of small blood vessels, which would increase the reflected wave returning to the heart. Augmentation pressure provides great insight into peripheral blood vessel changes and its relationship with autonomic balance. In our case, acute circadian misalignment increased augmentation pressure. Fortunately, our healthy participants did not have an increase in their blood pressure. The non-increase in blood pressure is likely caused by their healthy aortic blood vessel damping the effect of the reflected wave. If these poor sleep habits were to persist chronically, the risk of developing hypertension and related conditions would increase.

Unfortunately, because of scheduling issues, we have not yet analyzed any data regarding the collection of C-Reactive Protein (CRP). Results from this portion of the study are forthcoming.

Figure 5. Depicts the mean oxygen consumption (ml/kg/min) across three stages of the metabolic flexibility protocol between the aligned and misaligned sleep conditions.

Shifting toward the metabolic components of our study, we observed no difference in heart rate from rest to exercise under the misaligned condition compared with control. It does not appear that our protocol resulted in different heart rate responses to exercise across each stage. Congruently, there was no difference in oxygen consumption (Figure 5), fat oxidation, or carbohydrate oxidation. Taken collectively, these findings may suggest that while circadian misalignment is known to impair related physiological processes involved with our bodies’ changes during exercise, the healthy adult population may be protected from having these detriments impair their exercise performance. Since exercise is an environmental stressor to our body, taking it out of homeostasis, these healthy young adults may also be protected in resting conditions as well, sparing their cardiometabolic health.

 Interestingly, when assessing resting variables, ventilation exchange values (L/min) were lower in the misaligned intervention (Figure 6), while the respiratory exchange ratio remained unaffected. The observed increase in carbon dioxide production suggests a shift in cellular metabolism, resulting in higher resting ventilation rates. 

Figure 6. Depicts metabolic flexibility variables between the aligned and misaligned sleep conditions. * indicates statistical significance between the two conditions (p<0.05).

This metabolic change, along with signs of elevated sympathetic activity, indicates that our misalignment protocol affects both cellular energy processes and the body’s stress response system. Our early preliminary findings suggest that circadian misalignment does not influence resting or exercising oxygen consumption, fat oxidation, carbohydrate oxidation, or heart rate. However, we did observe a trend toward diminished fat oxidation during the misaligned protocol, which may prove to be significant as we collect more data. Further data collection, which we intend to conduct during the spring of 2025, is warranted to confirm the results of this preliminary study. We are hoping to reach twenty subjects.

Conclusion

The Summer Undergraduate Research Fellowship was an invaluable experience that came with many lessons to be learned. At the surface, the summer equipped us with many new skills used in an exercise physiology and wet lab, including blood pipetting, and anthropometric data collection such as height, weight, and body composition. Some of the exercise science-specific lab techniques included proctoring a submaximal exercise test and metabolic flexibility test using a metabolic cart, as well as collecting heart rate variability and blood pressure data. The data collection process was quite exciting, and working with human subjects was even more rewarding. Gaining new insight into data analysis, which we knew little about before conducting our study, was a fascinating process as well.

In preparing our manuscripts and abstracts for presentations, we were able to improve our scientific writing skills. The crucial thinking and problem-solving skills we advanced have enhanced our ability to approach complex problems logically in both personal and professional settings. By working as a collaborative team, we strengthened our ability to express our ideas clearly in an effective manner, which is essential for leadership and teamwork in research and other professional settings. We also tested our time management capacity, and improved our ability to prioritize tasks, manage multiple responsibilities, and meet deadlines while balancing data collection, analysis, and project deadlines effectively over the course of the summer.

This experience enhanced our readiness for our specific career goals as well. Gabe Dennett gained further excitement to pursue scientific research as a long-term career. He will attend a master’s program next fall and intends to pursue a PhD afterward as well, two goals solidified by such a successful SURF experience. For Nate Goodwin, this was a profoundly impactful experience that substantially grew his appreciation for scientific research. It also helped him realize that research is not something he wants to pursue as a career; instead, he will use this experience to propel a career in health care. For both, the gain in scientific knowledge and laboratory skills helped us feel empowered for graduate school and beyond.

We would like to first thank our mentor, Dr. Michael S. Brian, for his unrelenting support in our academic and research endeavors. This project would not have been possible without his willingness to mentor us or the opportunity to work alongside him in his lab throughout our time at UNH. Thank you to our willing participants for their time and efforts to see this study through to completion. This study would not have been possible without the help of other members of the cardiometabolic research group (Jack Souza, Sarah Guerrette, Kelly Brown), who assisted with the data collection and for whom we are truly grateful. Ultimately, we would like to thank the Hamel Center for Undergraduate Research for this incredible opportunity to gain research experience at this stage in our academic journey, and to Mr. Dana Hamel, the Class of 1959 Fund for Excellence, and the UNH Parents Association Undergraduate Research Fund for graciously funding our research.

References

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(left to right) Nate Goodwin, Michael Brian, and Gabe Dennett. Photo credit: Jeremy Gasowski

Author and Mentor Bios

Gabe Dennett, from Norton, Massachusetts, is pursuing a double major in exercise science and nutrition at the �鶹app. After graduating in May 2025, Gabe plans to enroll in the master’s program in exercise physiology at the University of Montana. He has been involved in campus housing at UNH as a Resident Assistant and Community Assistant and served as a student mentor at the Center for Academic Resources (CFAR). He is also a College of Health and Human Services Dean’s Ambassador, and an active member of the Association of Exercise Science Students.

Nathan Goodwin is an exercise science major and nutrition minor from Southwick, Massachusetts. He will graduate in May 2025 and plans to attend physical therapy school. Outside of research he is a Dean’s Ambassador and ACE mentor for the College of Health and Human Services, and a member of the Association of Exercise Science Students.

Michael S. Brian is an assistant professor in the Department of Kinesiology at UNH. Dr. Brian’s current research interests focus on the influence of exercise on twenty-four-hour blood pressure regulation, blood glucose regulation, and the impact of adiposity on vascular health in young adulthood.

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