Time of Day and Muscle Strength: A Circadian Output?

Abstract

For more than 20 years, physiologists have observed a morning-to-evening increase in human muscle strength. Recent data suggest that time-of-day differences are the result of intrinsic, nonneural, muscle factors. We evaluate circadian clock data sets from human and mouse circadian studies and highlight possible mechanisms through which the muscle circadian clock may contribute to time-of-day muscle strength outcomes.

Introduction

Many physiological processes in our body oscillate in a circadian, or daily manner, including core body temperature, heart rate, and blood pressure (6, 7, 12, 21, 47, 55, 74, 75, 107, 110). These daily rhythms in physiology are regulated by an intrinsic circadian system and are not just acute responses to cues from the environment. The contributions of the circadian system to skeletal muscle physiology are just starting to emerge with most research, to date, focused on the circadian pattern of skeletal muscle metabolism (24, 49, 67, 73). In this brief review, we suggest that human muscle strength exhibits a circadian pattern, supported by more than 20 years of research, demonstrating that maximum isometric strength varies over time of day. All of these studies report that muscle strength peaks in the late afternoon ∼1600–2000 h (22, 29–32, 44, 53, 113). The goals of this review are 1) to compile known data regarding time-of-day and muscle isometric strength, 2) review the research on two proposed extrinsic mechanisms, nerve-muscle activation and core temperature, that have been suggested to influence the time of day variation in human muscle strength, and 3) introduce concepts of the muscle circadian clock to discuss the potential for the intrinsic skeletal muscle clock as a contributor to observed time-of-day changes in maximal strength.

Time-of-Day Differences in Human Maximal Isometric Strength

For the purpose of this review, we have focused on maximal isometric strength, produced by tetanic contractions with no change in muscle length, as these measures provide a robust readout of the maximal force capabilities of skeletal muscle (111). All of these studies recorded measurements with a dynamometer, the most common instrument used to measure maximal isometric force or torque generated by the muscle (13, 80). In addition, we note that all of these studies were performed in human subjects, as we are not aware of published data in preclinical animal models. We purposely use the term “time of day” and not circadian here, as all the studies were all performed under normal light-dark conditions and, thus, do not conform to the requirement for constant environmental conditions with circadian studies (23).

We identified over 20 studies that have been performed to specifically assess time-of-day changes in isometric muscle strength (8, 16, 17, 22, 25, 29–32, 34, 44, 45, 53, 57, 63, 76, 79, 85, 86, 89, 90, 101, 113, 115). What was striking about all of these studies was the consistency of outcomes across several different muscle groups, showing maximal isometric muscle strength to be greatest in the late afternoon, (∼1600–2000) compared with morning (8, 16, 17, 22, 25, 29–32, 34, 44, 45, 53, 57, 63, 76, 79, 85, 86, 89, 90, 101, 113, 115) (FIGURE 1). It should be noted that these studies were primarily performed in cohorts of young, healthy males, so we cannot address potential sex or age differences. Of the 24 time-of-day strength studies that we reviewed for this paper, only five included female subjects. Additionally, the distribution of male-to-female subjects was never evenly distributed and ranged from 7 to 71% female inclusion (8, 30, 32, 53, 90). The typical age of subjects was between 21 and 35 yr of age, with two studies involving subjects younger than 21 (8, 89). Future studies, including different ages and both sexes, are required to better characterize time-of-day maximal isometric strength across the population.

FIGURE 1.

Conceptual cartoon illustrating maximal isometric strength as a circadian physiological output, raising the potential for muscle clock to modulate these changes

Some of the earliest studies in this field have reported a time-of-day difference in grip strength, conducting repeated measures in the morning and evening with the same cohort of subjects (9, 32, 84, 113). These studies found that there was a significant and reproducible ∼5–6% increase in grip strength in the evening. In a more recent study, Jasper et al. (44) performed grip strength measures every 3 h over a 24-h period identifying a peak in strength occurring ∼1800 h. Analysis of this more detailed time series data set shows that there was a 5.1% difference in grip strength between the highest to the lowest values, corroborating previous work, which demonstrated a similar time-of-day peak in grip strength, yet with lower temporal resolution (44). While grip strength was the earliest strength measure shown to exhibit time of day variations, the majority of the more recent research has focused on time-of-day strength measures of either the elbow extensor/flexors or the knee extensors/flexor muscle groups. Consistent with previous data from grip strength, these studies report very similar time of day increases in maximal isometric force and/or torque (16, 25, 29–31, 45, 57, 63, 79, 85, 86, 89, 115). More specifically, these studies report that the greatest force and/or torque measures occurred between 1700 and1800. Furthermore, these studies report that the morning-to-evening rise pattern in strength remains in larger muscle groups, such as the knee extensors/flexors, reporting an ∼4–13% increase. Differences in the magnitude of morning-to-evening increases in maximal isometric strength of the flexor/extensor muscle groups may, in part, be a result of differences in experimental protocols and standardized joint angles used to obtain these measures (61). For example, Gauthier et al. (29, 30) measured maximal isometric strength in elbow flexors, while maintaining the elbow at either 60° or 90° of flexion, and revealed greatest values of maximal isometric strength occurred around 1800, but the magnitude of the time-of-day difference was greater at 90° of flexion (7.63% increase) versus 60° flexion (4% increase). Similar to grip strength measures, all of these studies demonstrate that isometric strength of the extension/flexion muscles in the elbow and/or knee is greatest in the late afternoon, significantly increasing from morning to evening. Importantly, regardless of experimental protocol or muscle group, data from human time-of-day muscle performance all consistently report that maximal isometric strength demonstrates a robust morning-to-evening increase. This robust time-of-day difference in muscle strength strongly suggests that this functional change in skeletal muscle could be considered a circadian physiological output.

Possible Factors That May Contribute to Time-of-Day Muscle Strength

Muscle strength is well known to be modulated by patterns of neural recruitment and temperature. Thus, these two variables have been most studied as mechanisms underlying the morning versus afternoon strength differences. This section of the review will focus on the studies that have been performed to address potential mechanisms contributing to time of day strength differences.

We would note that there are other physiological variables with known time-of-day variations, such as heart rate and blood pressure, that could influence time-of-day maximal isometric strength. However, to date, there are no studies that have addressed their potential influence on time-of-day differences in isometric strength.

One of the most obvious mechanisms for differences in time-of-day muscle strength is the differential recruitment of motor units during the process of generating force (27). It has long been recognized that neural activation is a component of muscle strength, and strength improvements with training include aspects of neural adaptations (26). The potential for time of day neural recruitment has been approached, with two different experimental design strategies. One approach has used electromyography to measure muscle activation during time-of-day strength measures. The second approach used the interpolated twitch technique to override any upstream time-of-day differences with strength outcomes. The underlying hypothesis has been that time-of-day differences in maximal isometric strength would be due to time-of-day differences in either central drive/motivation and/or motor innervation patterns.

Several studies have been performed using electromyography as a readout of neural activation of muscle during strength testing in humans (30, 34, 53, 63, 86). Surface electromyography (sEMG) is a noninvasive technique used to indirectly gain insight into nerve-muscle activity during maximal isometric contractions (15, 39, 78, 108). It is largely agreed that sEMG data reflect the volume of muscle excitation and recruitment during isometric contractions (3, 43, 87, 108). Although there are concerns about the types of interpretations made with sEMG (82, 117), it is recognized as a reliable technique for isometric muscle contraction due to the curvilinear relationship that exists between sEMG amplitude and isometric force (108). Review of the time-of-day strength sEMG studies consistently report that even though time-of-day strength does change, there is no observable change in the pattern of neural recruitment over time of day (30, 34, 53, 63, 86). One outcome variable obtained from sEMG is the root mean square value of the EMG, used commonly to quantify the collective electrical activity of motor units during maximal voluntary isometric contractions (28, 87). Several studies have found that there is no significant time-of-day difference in root mean square values during maximal voluntary isometric contractions of either the knee and/or elbow extensor/flexor, or adductor pollicis muscles (30, 34, 53, 63, 86). These studies further report that, irrespective of no change in sEMG parameters across time of day, maximal isometric strength is consistently higher in the late afternoon.

The interpolation twitch technique provides extrinsic electrical stimulation coupled with maximal effort to assess the magnitude of motor unit recruitment during maximum isometric contractions. This approach has been used to ask whether there is a time-of-day difference in central drive or motivation, and if this might result in the morning versus afternoon strength differences observed. Martin et al. (53) used the interpolated twitch technique along with maximal voluntary contractions and found a similar morning-to-evening increase in maximal isometric strength in both voluntary and electrically induced muscle contractions. Of further note, during both the interpolated twitch and voluntary contraction protocols, the authors reported that the rate of force development and time to relaxation were greater in the late afternoon compared with the morning, parameters that are linked to intrinsic muscle properties, such as myosin type and calcium handling. Such work suggests further support that time-of-day differences in maximal isometric muscle strength are not due to changes in neural recruitment, but likely are due to the intrinsic properties of the muscle.

It is well documented that muscle force generation is temperature-sensitive in vivo, ex vivo, and in vitro (10, 18, 20, 36). Furthermore, it is well established that core body temperature exhibits an ∼1°C circadian rhythm with highest values during the afternoon (∼1700–1900) and lowest values occurring in the early morning (0400–0600) (6, 7, 47, 74–75). As a result, the correlation between time-of-day strength differences and core body temperature has been extensively studied, leading to the hypothesis that time-of-day isometric strength differences are regulated by core body temperature.

Previous work has reported that human muscle temperature in vivo can affect isometric force, demonstrating a 2% decrease in isometric force per 1°C decrease in muscle temperature. However, the range of temperatures studied (39°–30°C) was much larger than the daily change in core body temperature (10). In vitro and ex-vivo studies have shown a force-temperature relationship. Several studies report, across a range of experimental temperatures (1°–37°C, using different protocols) in isolated rodent muscle and myofibers that myofilament force production and calcium sensitivity are greater with an increased temperature (18, 20, 36, 72). Gauthier et al. (30) measured core temperature and maximal isometric strength at six timepoints throughout a 24-h day and found that changes in strength correlated with the changes in core body temperature. To address whether increasing core temperature is sufficient to improve morning strength, several studies have used passive heating of the subject using a warm room with and without specialized garments to elevate core body temperature. These studies have found that passive heating of core body temperature is not sufficient to increase morning maximal isometric strength (25, 60, 99, 102). Furthermore, Edwards et al. (25) used both passive heating with increased humidity before strength measures in the morning, and this did not improve morning strength either. In addition Edwards et al. reported that increasing core temperature through increased physical activity resulted in no significant increase in morning maximal isometric strength (25). The results of these studies indicate that time-of-day changes in core body temperature do not account for the time-of-day changes in maximal isometric strength. The findings from both neural recruitment and temperature studies suggest that time of day changes in maximal isometric force may be due to intrinsic changes within the muscles.

Could the Muscle Circadian Clock Influence Time of Day Strength?

The last component that we will review and suggest as a potential modifier of maximal isometric strength over time of day is the potential for the muscle circadian clock to effect intrinsic muscle properties that modulate strength. Increasingly, data in both humans and mice suggest that systemic or muscle-specific disruption of the circadian clock results in a reduction of maximal isometric muscle strength (5, 24, 51, 77, 83, 105). Studies in humans have shown that disruption of the circadian clock mechanism by sleep disruption or sleep deprivation using protocols of 36 h or more of interrupted sleep have been shown to significantly reduce maximal isometric strength by ∼17% (51, 77, 105). We note that among these studies, Vaara et al. (105) report no significant differences among EMG measures during maximal isometric contractions after sleep deprivation, yet additional studies are needed to fully characterize the effect of sleep deprivation on central motivation and its downstream consequences on isometric strength. Additionally, studies in genetic mouse models have shown that ablation or mutation of the core clock mechanism results in a ∼20% reduction of in vivo and ex vivo muscle force measurements (5, 24, 83).

Before starting to discuss potential sites through which the muscle circadian clock might modulate time-of-day muscle strength, a general description and introduction is needed of the circadian clock mechanism itself. In mammals, the circadian clock exists in virtually every cell in the body and is a transcription-translation feedback loop that takes ∼24 h (46, 65, 97). The molecular components of the circadian clock include the core clock genes Bmal1, Clock, Period 1/2, and Cryptochrome 1/2 (46, 65, 97). Beyond its role as a time-keeping mechanism, the circadian clock is now recognized as a regulator of daily, rhythmic pattern of gene expression, modulating expression of thousands of genes over a 24-h period, and commonly referred to as clock output. Recent research demonstrates that clock output is highly tissue-specific (2, 5, 56, 64, 66, 83, 91, 116) and that skeletal muscle has its own intrinsic circadian clock output, as has been described for many other tissues (5, 54, 66, 83, 116). It is also important to note that analysis of clock genes has been performed with human muscle, and the pattern of clock gene expression is similar between diurnal humans and nocturnal rodents, with the peak of Bmal1 gene expression occurring at the transition from the active to the rest phase of the day (35).

The goal of the section below is to discuss three different genes that we selected following bioinformatic analysis of circadian transcriptome data sets available from both human and mouse muscle that could affect muscle strength properties in a time-of-day manner (4, 54, 66). These specific examples were selected on the basis of the following criteria: 1) the genes demonstrate rhythmic expression in publicly available mouse and/or human skeletal muscle circadian transcriptomic data sets, and 2) the genes have well-established roles in regulating muscle contraction and force (4, 54, 66, 92, 95, 109), and 3) the genes exhibit changes in expression with mouse models in which the clock is disrupted only in skeletal muscle (24, 38, 83). This last category is important, as these mice have an intact central clock and still exhibit normal circadian behaviors, such as cage activity, so altered gene expression in the muscle is more likely to be due to clock disruption and less likely to be downstream of either daily feeding or activity patterns. The three examples that we have chosen, fall within the categories of calcium regulation, kinases that target contractile proteins, and sarcomere-associated proteins. It is important to note that these genes are simply meant as conceptual examples to illustrate how the skeletal muscle clock may contribute to time-of-day differences in muscle strength.

The first example was selected following functional cluster analysis from circadian transcriptome data and was from the “Calcium Signaling Pathway” (FDR of 0.036: Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8) (41, 42, 54). Within this cluster, we identified calmodulin genes from both the human and mouse data sets (54, 66). There are three isoforms of calmodulin, Calm1/2/3, and Perrin et al. (66) report that Calm1 and Calm2 mRNA are rhythmically expressed in human skeletal muscle. Analysis of mouse muscle in which the core clock gene, Bmal1, is ablated in skeletal muscle found that Calm3 mRNA expression in skeletal muscle was significantly decreased compared with wild type (54), suggesting that the expression of Calm3 is modulated by the skeletal muscle clock mechanism. Calmodulin (CaM) is a small protein that is known to interact with many different target proteins in a calcium-dependent manner in skeletal muscle (109). For example, skeletal muscle myosin light-chain kinase is a known modulator of skeletal muscle force potentiation, and its regulatory role is dependent on both CaM and calcium (52, 58, 117). Additionally, studies have demonstrated that in muscle, CaM can physically interact with the calcium release channel, the ryanodine receptor, in the sarcoplasmic reticulum (69, 88, 109). The interaction of CaM with the ryanodine receptor has been shown to regulate the amount of time in which the calcium channel remains open, effectively acting as a physiological rheostat for calcium release into the sarcoplasm (68, 88). Thus, these data suggest the possibility that differential expression of CaM over time of day could regulate the function of the ryanodine receptor, leading to modulation of calcium release kinetics, influencing a downstream effect on muscle strength.

The second example that we highlight belongs to the functional cluster “Phosphorylation” (FDR of 0.058) (41, 42, 54). Phosphorylation of muscle contractile proteins and its impact on force are a topics with a long research history (93, 96, 106). The most well-known link between phosphorylation and muscle strength is via phosphorylation of the myosin regulatory light chain (MLRS), also known as myosin light chain 2 (94, 98, 103, 114, 117). Several studies have shown that changes in myosin light-chain phosphorylation are directly linked to altered force production of myofilaments both in vitro as well as in vivo (93, 103, 117). Furthermore, circadian transcriptome analysis reveals that skeletal muscle myosin light-chain kinase (MLCK) is one of the genes that is expressed downstream of the skeletal muscle circadian clock (4, 54). MLCK mRNA is rhythmically expressed in vitro in cultured mouse myotubes (4) and in vivo in human skeletal muscle (66). Further, MLCK mRNA expression is downregulated by ∼19% in skeletal muscle-specific Bmal1 knockout mice muscle tissue compared with wild-type (54). A recent paper focused on time-of-day muscle strength with phosphoproteome analysis of muscle biopsies taken immediately following the strength measures (1). Although they did not detect a time-of-day difference in myosin light-chain phosphorylation, they did detect altered phosphorylation of several proteins within the sarcomere at the M line, including Myomesin 2 (1). To date, the upstream kinase(s) that target those sites are not known, but the results of this study highlight the potential contribution of contractile protein phosphorylation as a potential mechanism modulating observed time-of-day strength measures.

Lastly, the human and mouse muscle circadian transcriptome data have identified the well-known muscle-specific transcription factor, MyoD1, as a circadian gene (38, 54, 66). With its known role in regulating muscle-specific genes (5, 19, 38, 100), the skeletal muscle circadian clock is linked with genes critical for sarcomere structure and function. One example gene is Telethonin or Titin-Cap (Tcap) (38, 54). TCAP is a Z-line protein that has a known functional role as a “glue” anchoring the NH₂-terminal ends of Titin molecules within the Z line (11, 48, 81). A recent study by Swist et al (95) has demonstrated that the loss of Z-line anchoring of Titin results in loss of sarcomere integrity and results in significant force decrements in both passive and calcium-activated tension of isolated mouse myofibers. Additionally, loss-of-function mutations in TCAP play a causative role for Limb Girdle Muscular Dystrophy (or LGMD 2G), which is characterized by progressive limb-girdle skeletal muscle weakness (14, 59, 62). Tcap mRNA has been shown to be rhythmically expressed in skeletal muscle, protein levels also vary over time of day (112), and skeletal muscle-specific ablation of Bmal1 in mice results in a greater than 50% decrease in mRNA expression (54, 68, 70). Taken together, the expression of Tcap is regulated directly by the skeletal muscle circadian clock in muscle, with a 30% difference in levels from the end of the rest phase to the end of the active phase. TCAP is an important component of the Z line with links to functional outcomes via its association with Titin (11, 33, 37, 40, 50, 71, 104). This makes it an interesting potential target for modulating daily variations in skeletal muscle strength.

We do want to note that the above discussion is largely focused on changes in gene expression at the mRNA level, as there is not much data about time-of-day differences in protein levels. Future studies will require combining either targeted protein expression or more unbiased proteomic approaches with careful circadian design to identify potential molecular sites within muscle that contribute to time-of-day differences in strength. This suggested role for the skeletal muscle clock provides exciting avenues for future studies to better understand time-of-day modulation of skeletal muscle function and physiology.

Conclusions

In humans, skeletal muscle strength has been shown to vary over time of day with greatest strength values occurring during the late afternoon ∼1600–2000 h. This has been robustly demonstrated with smaller muscles that are involved in grip strength, to upper limb muscles that are involved in elbow strength, and the large muscle groups of the leg that regulate strength around the knee. Although the changes in muscle strength over time of day are very robust, to date, the mechanisms contributing to this circadian physiology are not well defined. Analysis of EMG with maximal voluntary contractions or with interpolated twitch methods strongly suggest that the time-of-day differences observed in muscle strength are not due to neural activation over time of day. Analyses of core body temperature as a potential factor modulating muscle strength over time of day have utilized passive warming approaches, and these studies demonstrate that increased temperature alone is not sufficient to account for the improved maximal isometric muscle strength in the afternoon. Lastly, we review the concept of the skeletal muscle circadian clock as a potential modulator of muscle strength. We have provided three examples from circadian gene expression data sets from human and mouse muscles. While it is too early to make any conclusions about the potential links between the muscle circadian clock and time of day differences in maximal isometric strength, we suggest that future studies take into account this intrinsic mechanism within the muscle and consider ways in which it may contribute. We can envision many complementary studies, including properly controlled human circadian muscle biopsies with additional downstream protein and/or proteomic analyses, which will be required to help understand the changes in muscle strength over time of day.