Circadian rhythms are fundamental daily cyclical processes influencing various physiological and behavioral aspects, exhibiting considerable inter-individual variation. Chronotype, or circadian preference, reflects an individual's inherent predisposition towards earlier or later sleep-wake cycles, stemming from the interplay between internal circadian rhythms and sleep needs. While the influence of circadian rhythms on basic physiological processes (e.g., cell cycle, body temperature, sleep-wake cycle) is well-established, the impact of chronotype on human brain physiology and cognition remains less understood. This research gap is particularly significant given the increasing detachment of modern lifestyles from the natural 24-hour day-night cycle. A comprehensive understanding of chronotype's influence on brain function and cognitive performance holds considerable implications for various aspects of human well-being, including public health, workplace productivity, educational outcomes, and the pathophysiology of various diseases. This study investigates the interplay between chronotype and time-of-day on several key aspects of human brain physiology, specifically cortical excitability and neuroplasticity. These physiological processes underpin adaptive behavior in both healthy individuals and clinical populations. The research also examines the relationship between chronotype and performance on motor learning tasks and higher-order cognitive functions such as attention and working memory, exploring the underlying mechanisms that drive chronotype-dependent performance differences. Technological advancements in neuroscience, particularly non-invasive brain stimulation (NIBS) techniques such as transcranial magnetic stimulation (TMS) and transcranial electrical stimulation (tES), provide valuable tools for investigating and modulating brain function. TMS, based on electromagnetic induction, allows for the non-invasive measurement of various aspects of cortical excitability, providing insights into the neurotransmitter systems involved (e.g., glutamatergic, dopaminergic, GABAergic, cholinergic). tDCS, a form of tES, utilizes weak electrical currents to modulate cortical excitability, inducing long-term potentiation (LTP)-like or long-term depression (LTD)-like plasticity. These plasticity processes are considered crucial substrates for learning and memory formation. While animal studies have demonstrated a significant circadian impact on hippocampal plasticity and LTP, the role of circadian preference in modulating human cortical excitability and cognitive functions, including plasticity and learning, requires further investigation. This study systematically explores this interaction, aiming to advance our understanding of human brain function and its implications for daily life.

A substantial body of research highlights the significant influence of circadian rhythms on various biological processes. Studies focusing on molecular and cellular mechanisms have uncovered intricate links between circadian rhythms and physiological functions in mammals, including humans. In recent years, research interest has expanded to include the impact of circadian rhythms on brain physiology and cognition, facilitated by advances in cognitive neuroscience. This renewed interest stems from the growing recognition of the pervasive influence of circadian rhythms on human behavior and well-being, particularly in the context of modern lifestyles that frequently disrupt the natural 24-hour cycle. Studies have demonstrated the impact of circadian rhythms on cortical excitability, suggesting a direct link between internal biological clocks and brain function. However, research specifically addressing the role of chronotype in these processes has been limited. Existing literature indicates a strong circadian influence on hippocampal plasticity and LTP in animal models. Furthermore, research suggests that neural excitability, both in invertebrates and the human motor cortex, is modulated by circadian rhythms. However, the extent to which circadian preference affects human cortical excitability, neuroplasticity, and consequently cognitive functions, remains largely unexplored. This study aims to fill this knowledge gap, contributing to a more complete understanding of the intricate relationship between chronotype, brain physiology, and cognition.

This study employed a multi-faceted approach involving non-invasive brain stimulation (NIBS) techniques, behavioral tasks, and electroencephalography (EEG) recordings to investigate the influence of chronotype on human brain physiology and cognition. Thirty-two healthy young adults (16 females, mean age 26.62 ± 5.01 years), classified as either early chronotypes (ECs) or late chronotypes (LCs) based on the Morningness-Eveningness Questionnaire (MEQ), participated in the study. Chronotype classification focused on moderate early and late chronotypes to minimize variability in sleep timing and duration. The sample size was determined a priori based on power analyses. **Cortical Excitability Monitoring with TMS:** Various single-pulse and paired-pulse TMS protocols were used to assess corticospinal and intracortical excitability within the motor cortex. These protocols measured parameters such as resting motor threshold (RMT), active motor threshold (AMT), input-output (I/O) curves, short-interval intracortical inhibition (SICI), intracortical facilitation (ICF), I-wave facilitation, and short-latency afferent inhibition (SAI). Measurements were conducted at both circadian-preferred and non-preferred times (morning and evening sessions, counterbalanced across groups). **Neuroplasticity Induction with tDCS:** Transcranial direct current stimulation (tDCS) was applied to the primary motor cortex to induce LTP-like or LTD-like plasticity. Participants underwent six tDCS sessions (anodal, cathodal, and sham at both morning and evening time points) with a 1-week interval between sessions. MEP measurements were taken before, during, and after tDCS to assess neuroplastic effects. **Behavioral Measures:** To evaluate motor learning, participants performed a serial reaction time task (SRTT), measuring reaction time (RT) and accuracy. Cognitive functions were assessed using the 3-back letter task (working memory), the Stroop color-word task (selective attention), and the AX-continuous performance test (AX-CPT) (sustained attention). These behavioral tests were administered during EEG recording at both circadian-preferred and non-preferred times. **EEG Recording and Analysis:** EEG data were recorded continuously during cognitive task performance. Data were preprocessed to remove artifacts, and event-related potentials (ERPs) were extracted and analyzed for various components (P300, N200, N450) to identify electrophysiological correlates of cognitive processes. Resting-state EEG data were also analyzed to assess theta and alpha oscillations as indirect markers of sleep pressure. **Statistical Analysis:** Mixed-model ANOVAs with repeated measures were primarily used to analyze the data, with post-hoc tests employed for pairwise comparisons. Correlational analyses (Pearson's correlation) were used to examine relationships between neuroplasticity, motor learning, and cognitive performance.

This study yielded several significant findings related to the association between chronotype and various aspects of brain physiology and cognition: **Enhanced Cortical Excitability at Circadian-Preferred Times:** Both early and late chronotypes exhibited enhanced corticospinal excitability, increased cortical facilitation, and reduced cortical inhibition at their circadian-preferred times. This suggests a chronotype-dependent modulation of neurotransmitter systems (glutamatergic and GABAergic) influencing cortical excitability. **Chronotype-Dependent Neuroplasticity:** LTP/LTD-like plasticity in the motor cortex was significantly greater at the circadian-preferred time in both early and late chronotypes, indicating that the capacity for inducing neuroplastic changes is influenced by the individual's chronotype and the time of day. **Superior Performance on Motor Learning and Cognitive Tasks at Circadian-Preferred Times:** Both early and late chronotypes demonstrated significantly better performance on motor sequence learning (SRTT) and cognitive tasks (working memory, selective attention, and sustained attention) at their circadian-preferred times. This enhanced performance was accompanied by larger amplitudes of task-relevant ERP components (P300, N200, N450), suggesting that chronotype modulates electrophysiological aspects of information processing. **Correlation between Neuroplasticity and Motor Learning:** A positive correlation was found between the effects of anodal tDCS (LTP-like plasticity) and motor sequence learning in late chronotypes during their circadian-preferred time (evening). This association supports a functional link between plasticity and performance in specific chronotype and time-of-day conditions. **No Significant Differences in Sleepiness and EEG Markers of Sleep Pressure:** No significant differences in subjective sleepiness ratings (KSS) or resting EEG theta oscillations (indirect markers of sleep pressure) were observed between chronotypes or across the morning and evening sessions. This indicates that the observed differences in brain physiology and cognition are not solely attributable to sleep pressure differences between the groups and time points.

The findings of this study provide compelling evidence for the association between chronotype and various aspects of human brain function and cognition. The observed enhancements in cortical excitability, neuroplasticity, and cognitive performance at circadian-preferred times strongly suggest that chronotype significantly modulates neurotransmitter systems (glutamatergic and GABAergic) and their influence on cortical plasticity and cognitive processes. The consistency of these effects across both early and late chronotypes highlights the fundamental role of circadian rhythms in shaping individual differences in brain function and cognitive abilities. The study's findings have important implications for our understanding of the interaction between internal biological clocks and brain plasticity. The significant influence of chronotype on both basic physiological mechanisms and higher-order cognitive processes underscores the need to consider individual circadian preferences when designing and interpreting studies involving brain stimulation techniques or cognitive assessments. The observed differences in tDCS-induced neuroplasticity highlight the potential of optimizing NIBS interventions by tailoring their timing to individual chronotypes. Moreover, the study's results support the optimization of educational and work schedules to maximize cognitive performance by aligning activities with individuals' circadian preferences.

This study demonstrates a strong association between chronotype and both brain physiology and cognitive performance. Both early and late chronotypes show enhanced cortical excitability, neuroplasticity, and performance on motor learning and cognitive tasks at their circadian-preferred times. These findings suggest that chronotype is a significant factor to consider in research involving brain stimulation techniques and cognitive assessments, with potential implications for optimizing interventions and scheduling to improve performance and well-being. Future research should focus on clarifying the interaction between chronotype, sleep pressure, and cognitive performance using more precise measures of sleep parameters, and investigating the specific molecular mechanisms underlying these chronotype-dependent differences in brain function.

The findings of this study should be interpreted cautiously, acknowledging certain limitations. First, the observed effects might not be exclusively attributed to chronotype, as the study design included fixed measurement times that may have inadvertently influenced sleep pressure levels differently across groups. While the researchers controlled for some sleep-related variables (sleep duration, sleepiness ratings, and resting EEG markers), the use of direct and objective measures of sleep parameters (e.g., polysomnography) would have provided a more comprehensive assessment of sleep pressure effects. Second, the motor cortex served as the primary focus of physiological measurements, making it challenging to definitively extrapolate these findings to other brain regions. While the cognitive tasks assessed involved higher brain regions, further research using more widespread brain imaging techniques (e.g., fMRI) is needed to fully elucidate the extent of the chronotype-dependent effects across the brain. Finally, despite efforts to control for extraneous variables, some uncontrolled factors might still have influenced the results. Although the sample size of this study was calculated and determined a priori based on power analysis, it remains limited to young adults, making it difficult to generalize the findings to other age groups.