1. Introduction {#sec0005}
===============

In space, microgravity affects the central circulation in humans and induces a number of adaptive changes within the cardiovascular system. Previous investigations showed that the baroreflex sensitivity fluctuates along with altered blood volume distribution \[[@bib0005], [@bib0010], [@bib0015]\], which affects neural mechanisms involved in dynamic cardiovascular coordination. Several reports indicate that heart rate is maintained at preflight values \[[@bib0020], [@bib0025], [@bib0030]\] and that parasympathetic activity is reduced [@bib0020] in space. Cardiac output and stroke volume are reportedly increased in space as a result of an increase in preload to the heart induced by upper body fluid shift from the lower body segments with no major difference in sympathetic nerve activity [@bib0030]. However, high sympathetic nervous activity, measured invasively by microneurography in peroneal nerves, has been simultaneously detected in space in three astronauts [@bib0035] compared to the ground-based supine posture. Physiologic acclimation to space flight is a complex process involving multiple systems [@bib0040]. How the neural cardiovascular coordination adapts to the space environment is still poorly understood in humans.

When faced with a new environment, humans must first acclimate to it in order to survive. This includes the cardiovascular system. Adjustment to the new environment to improve quality of life follows, involving the autonomic, endocrine and immune systems, among others. But, as we reported previously [@bib0045], the "intrinsic" cardiovascular regulatory system, reflected by the fractal scaling of HRV \[[@bib0045], [@bib0050], [@bib0055]\], did not adapt to the new microgravity environment in space during long-duration (about 6-month) spaceflights. By contrast, after 6 months in space, the circadian rhythm of heart rate had adapted to the new microgravity environment in space [@bib0060], an important observation since disruption of circadian rhythms adversely affects human health \[[@bib0065], [@bib0070]\]. As humans plan for long-term space exploration, it is critical to ascertain that the regulatory system can function well in a microgravity environment.

The power-law fractal scaling of heart rate variability (HRV) relates to the autonomic [@bib0075], endocrine [@bib0075], immune, inflammatiory \[[@bib0080], [@bib0085]\], mental, cognitive [@bib0090], and behavioral systems, which operate at multiple frequency ranges, from the 1 Hz cardiac cycle to circadian and even secular variations, as part of a broad time structure, the chronome [@bib0095]. Herein, we examine how the space environment affects HRV in specific frequency regions, broken down into 8 different frequency ranges. We focus on the basic rest-activity cycle (BRAC), well known since Kleitman [@bib0100], who showed regularly occurring alternations between non-REM and REM (Rapid Eye Movement) sleep. The BRAC is involved in the functioning of the central nervous system and manifests time-dependent changes in human performance, including oral activity cycles (e.g., eating, drinking, smoking).

2. Methods {#sec0010}
==========

2.1. Subjects {#sec0015}
-------------

Ten healthy astronauts (8 men, 2 women) participated in this study. Their mean (± SD) age was 49.1 ± 4.2 years. Their mean stay in space was 171.8 ± 14.4 days. On the average, astronauts had already experienced spaceflight 0.9 ± 0.7 times and had passed class III physical examinations from the National Aeronautics and Space Administration (NASA). This study obtained consent from all subjects and gained approval from the ethics committee jointly established by the Johnson Space Center and Japan Aerospace Exploration Agency (JAXA). A detailed explanation of the study protocol was given to the subjects before they gave written, informed consent, according to the Declaration of Helsinki Principles.

2.2. Experimental protocols {#sec0020}
---------------------------

Ambulatory around-the-clock 24-hour electrocardiographic (ECG) records were obtained by using a two-channel Holter recorder (FM-180; Fukuda Denshi). Measurements were made five times: once before flight (Control), three times during flight (International Space Station (ISS) 01, ISS02, and ISS03), and once after return to Earth (After flight). The before-flight measurement session (Control) was conducted on days 234.4 ± 138.4 (63 to 469) before launch in all but one astronaut who had technical problems with his before-flight record. In his case, a replacement control record was obtained 3.5 years after return to Earth. The three measurement sessions during flight were taken on days 20.8 ± 2.9 (18 to 28, ISS01), 72.5 ± 3.9 (67 to 78, ISS02) and 152.8 ± 16.1 (139 to 188, ISS03) after launch, the latter corresponding to 19.1 ± 4.1 days (11 to 27) before return (ISS03). The last measurement session was performed on days 77.2 ± 14.4 (37 to 127 days) after return to Earth (After flight).

2.3. Analysis of heart rate variability and measurement of 1/f fluctuations in HR dynamics {#sec0025}
------------------------------------------------------------------------------------------

The measurement procedures and data collection were conducted as previously reported \[[@bib0045], [@bib0060]\]. Briefly, for HRV measurements, QRS waveforms were read from continuous electrocardiographic (ECG) records. The RR intervals between normal QRS waveforms were extracted as the normal-to-normal (NN) intervals. The measured NN intervals were A/D converted (125-Hz) with 8-ms time resolution. After the authors confirmed that all artifacts were actually removed and that the data excluded supraventricular or ventricular arrhythmia, frequency-domain measures [@bib0075] were obtained with the MemCalc/CHIRAM (Suwa Trust GMS, Tokyo, Japan) software [@bib0105].

Time series of NN intervals covering 5-min intervals were processed consecutively, and the spectral power in different frequency regions was computed, namely in the "high frequency (HF)" (0.15--0.40 Hz; spectral power centered around 3.6 sec), "low frequency (LF)" (0.04--0.15 Hz; spectral power centered around 10.5 sec), and "very low frequency (VLF)" (0.003--0.04 Hz; 25 sec to 5 min) regions of the Maximum Entropy Method (MEM) spectrum. VLF power was further broken down into "VLF band-1" (0.005--0.02 Hz; 50 sec to 3.3 min), "VLF band-2" (0.02--0.03 Hz; 33 to 50 sec) and "VLF band-3" (0.03--0.15 Hz; 6.7 to 33 sec).

Time series of NN intervals were also processed consecutively in 180-min intervals, progressively displaced by 5 min, to estimate the "ultra-low frequency" (ULF) component (0.0001--0.003 Hz; periods of 2.8 hours to 5 min), further broken down into: "ULF band-1" (0.0001--0.0003 Hz; 166.7 to 55.5 min), "ULF band-2" (0.0003--0.001 Hz; 55.5 to 16.6 min), and "ULF band-3" (0.001--0.005 Hz; 16.6 to 3.3 min). Thus, 8 different frequency regions were examined: "HF", "LF", "VLF01", "VLF02", "VLF03", "ULF01", "ULF02", and "ULF03". Results representing each HRV component were averaged over the entire 24-hour.

To evaluate the 1/f^β^-type scaling in HRV, the log~10~\[power\] (ordinate) was plotted against log~10~\[frequency\] (abscissa) and a regression line fitted to estimate the slope β, as reported earlier [@bib0045]. Focus was placed on the frequency range of 0.0001--0.01 Hz (periods of 2.8 hours to 1.6 minutes), as previously reported [@bib0045].

2.4. Fit of 4-component cosine model {#sec0030}
------------------------------------

A multiple-component model consisting of cosine curves with anticipated periods of 24, 12, 8 and 1.5 hours was fitted to various HRV endpoints by cosinor [@bib0110] to assess their time structure and to determine how the latter may have been modified in space. The model includes the usually prominent circadian rhythm (24-hour period) and its first two harmonic terms with periods of 12 (circasemidian) and 8 (circaoctohoran) hours, as well as the BRAC (with a period of about 90-min). Using a (least squares) regression approach, the cosinor does not require the data to be equidistant, and can thus handle missing values in cases when artifacts prevented the computation of HRV endpoints in some of the 5-min or 180-min intervals. Analyses considered primarily the Midline Estimating Statistic Of Rhythm (MESOR, a rhythm-adjusted mean) and the amplitude of each of the 4 components, as a measure of the extent of predictable change within each cycle. The 4-component model was fitted to 24-hour records of NN intervals, total power (TF), and power in the ULF (separately also in the ULF01, ULF02, and ULF03), VLF, LF, and HF regions of the MEM spectrum.

2.5. Inter-individual differences in HRV response to microgravity {#sec0035}
-----------------------------------------------------------------

Consistent differences in various HRV endpoints were noted in the way astronauts responded to microgravity. Examination of the inter-individual differences prompted the classification of the 10 astronauts into 2 clearly distinct groups. Hence, the influence of the space environment was also assessed separately in each group.

2.6. Statistical analyses {#sec0040}
-------------------------

Since we previously showed that the fractal scaling of HRV did remain altered in space as compared to Earth during long-term (∼ 6-month) spaceflights, this study specifically examines the behavior of HRV in 8 different frequency regions of the spectrum (ULF01, ULF02, ULF03, VLF01, VLF02, VLF03, LF, and HF), which can be considered to provide independent information. Adjustment for multiple testing thus uses a P-value of 0.05/8 to indicate statistical significance, using Bonferroni\'s inequality to adjust for multiple testing. The same correction is applied to other HRV endpoints shown for the sake of completeness, noting the high degree of correlation existing among different indices. We test whether HRV endpoints differ between space and Earth while showing no change among the 3 records obtained in space.

In order to do so, estimates of HRV endpoints averaged over 24 hours were expressed as mean ± SD (standard deviation). To minimize inter-individual differences in HR and HRV among the 10 astronauts that may obscure an effect of the space environment, 24-hour mean values of each variable were expressed as a percentage of mean, computed across the 5 sessions (before flight, ISS01, ISS02, ISS03, and after return to Earth) contributed by each astronaut. In this way, astronauts serve as their own longitudinal control. The two-sided paired-t and one-way analysis of variance (ANOVA) for repeated measures were applied on these relative values for the space vs. Earth difference and for comparing the 3 records in space, respectively.

Estimates of the MESOR and of the relative amplitude of each of the 4 anticipated components (with periods of 24, 12, 8, and 1.5 hours, expressed as a percentage of MESOR) of the selected HRV endpoints were considered as imputations for a comparison of HRV endpoints obtained during ISS03 versus before-flight. The statistical significance of change between the two sessions was determined using the 2-tailed paired t test. Inter-group differences were determined using the two-tailed Student t-test. P-values less than 0.05, adjusted for multiple testing according to Bonferroni\'s inequality, were considered to indicate statistical significance. The Stat Flex (Ver. 6) software (Artec Co., Ltd., Osaka, Japan) was used.

3. Results {#sec0045}
==========

3.1. Change in time structure of heart rate variability during long-duration spaceflight {#sec0050}
----------------------------------------------------------------------------------------

Average HRV endpoints during each of the 5 sessions are shown in [Table 1A](#tbl0005){ref-type="table"}. Results from a comparison of their relative values between space and Earth and across the 3 sessions on the ISS are summarized in [Table 1B](#tbl0010){ref-type="table"}. On average, among the 10 astronauts, no differences were found in HR (or NN) or in SDNN, the standard deviation of NN intervals. As reported earlier, the fractal scaling of HRV (slope β) was statistically significantly less steep in space than on Earth, while no changes were observed across the 3 records obtained in space, [Table 1A](#tbl0005){ref-type="table"}, [Table 1B](#tbl0010){ref-type="table"}. This result may be accounted for by the large space-Earth difference observed in the ULF frequency region of the spectrum, which is statistically significant for ULF02 and ULF03, as well as for ULF01 once it is normalized by the total spectral power (TF). These HRV endpoints did not differ among the 3 sessions recorded on the ISS, [Table 1A](#tbl0005){ref-type="table"}, [Table 1B](#tbl0010){ref-type="table"}. Of all the HRV endpoints considered herein, apart from β and the spectral power in the 3 ULF bands, only SDmean5 and SDmean30 show a lasting difference in space as compared to Earth, [Table 1A](#tbl0005){ref-type="table"}, [Table 1B](#tbl0010){ref-type="table"}.

Differences in β and the spectral power in the 3 ULF bands may stem from changes occurring around a frequency of one cycle in about 90 min. Indeed, β is computed over a frequency range centered around one cycle in about 90 min (1.7--166 min). Its absolute value decreased from 1.087 ± 0.130 (control, before flight) to 0.924 ± 0.095 (ISS03) (p \< 0.01). Correspondingly, ULF01/TF, also centered around 90 min, increased from 0.207 ± 0.053 to 0.310 ± 0.090, whereas ULF02/TF and ULF03/TF decreased from 0.189 ± 0.037 to 0.136 ± 0.030 and from 0.219 ± 0.035 to 0.151 ± 0.034, respectively.

3.2. Individual HRV response to microgravity associated with change in parasympathetic nerve activity {#sec0055}
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Individual 24-hour records of NN intervals (and hence instantaneous HR values) showed striking differences among the 10 astronauts. In 7 of them (Group 1), the 24-hour standard deviation (SD) of NN intervals was much lower (74.7--105.4 msec) than in the other 3 (Group 2) (171.7--196.0 msec) (Student t = 10.462, p \< 0.001). The two groups also differed in their average NN intervals (820. 8 ± 44.6 vs. 1023.2 ± 54.2, Student t = 2.610, p = 0.031). The inter-group difference in SD (NN) persisted during ISS01 (t = 3.451, p = 0.009), ISS02 (t = 4.615, p = 0.002), and ISS03 (t = 3.430, p = 0.009), as well as after return to Earth (t = 3.287, p = 0.011), when a difference in average NN intervals was also observed (t = 2.610, p = 0.031). Moreover, astronauts in Group 1 tended to respond to the space environment by increasing their average NN interval (decreasing their HR). The inter-group difference in response was statistically significant during ISS02 (t = 2.814, p = 0.023) and ISS03 (t = 3.515, p = 0.008), when the average NN intervals of all 7 astronauts of Group 1 was increased (on average by 85.4 ± 59.0 msec, t = 3.825, p = 0.009) and that of all 3 astronauts of Group 2 was decreased (on average by 41.9 ± 23.6 msec, t = 3.072, p = 0.092). [Table 2](#tbl0015){ref-type="table"} lists individual results during each of the 5 recordings, illustrating strong inter-individual differences in the HRV response to the space environment.

3.3. Power-law scaling β and ULF component of astronauts whose heart rate decreased in space {#sec0060}
--------------------------------------------------------------------------------------------

As seen for all 10 astronauts, the absolute value of β was also statistically significantly decreased in space (ISS03: 0.944 ± 0.097) as compared to preflight (1.144 ± 0.102) for the 7 astronauts of Group 1. Their ULF02 and ULF03 power was statistically significantly decreased from 915.0 ± 320.4 msec^2^ to 673.6 ± 275.3 msec^2^ and from 1017.4 ± 268.1 msec^2^ to 647.6 ± 192.5 msec^2^, respectively. In Group 2, there were no statistically significant differences in any of the HRV endpoints.

3.4. Change in chronome components (notably the basic rest-activity cycle) of heart rate variability during long-duration exposure to microgravity in space {#sec0065}
-----------------------------------------------------------------------------------------------------------------------------------------------------------

Changes during the 6-month spaceflight in the relative amplitudes of the 24-, 12-, 8-, and 1.5-hour components, expressed as a percentage of the MESOR, are shown in [Table 3](#tbl0020){ref-type="table"} for NN intervals, β, TF, and the different frequency ranges of the spectrum. On the average, the 90-min amplitude of TF, ULF and ULF01 increased 2- to 3-fold in space in astronauts of Group 1, whereas it decreased in those of Group 2, [Table 3](#tbl0020){ref-type="table"}. During ISS03 as compared to preflight, the BRAC amplitude of TF increased from 154.9 ± 105.0 to 532.7 ± 301.3 msec^2^, or from 3.2 to 11.3% of MESOR (n = 7), that of ULF increased from 117.9 ± 57.5 to 442.4 ± 202.9 msec^2^, or from 4.1 to 15.8% of MESOR (n = 7) and that of ULF01 increased from 124.3 ± 82.8 to 427.6 ± 214.8 msec^2^, or from 8.9 to 31.2% of MESOR (n = 7). In astronauts of Group 2, the 90-min amplitude of ULF01 decreased from 801.6 ± 155.6 before flight to 452.0 ± 239.9 during ISS02, or from 30.8 to less than 20% of the MESOR in space (n = 3), [Table 3](#tbl0020){ref-type="table"}.

Two examples of the fitted model to the TF data are shown in [Fig. 1](#fig0005){ref-type="fig"}, comparing the record during ISS03 (right) with the preflight record (left). In one case ([Fig. 1](#fig0005){ref-type="fig"}A), the 90-min amplitude increased from 59.5 to 684.5 msec^2^, with practically no change in the circadian amplitude. In another case ([Fig. 1](#fig0005){ref-type="fig"}B), the 90-min amplitude also increased from 71.4 to 754.5 msec^2^, but it was accompanied by an increase in the 24-hour amplitude from 529.8 to 3196.4 msec^2^.

3.5. Implications of heart rate response to space environment for adaptation to microgravity {#sec0070}
--------------------------------------------------------------------------------------------

To better understand the meaning of a difference in HRV response to the space environment, we compared the characteristics of the 4-component model fitted to some HRV endpoints before flight and during ISS03 between Groups 1 and 2. Before flight, the MESOR of TF, ULF and VLF spectral power was statistically significantly lower, on average, in astronauts of Group 1 as compared to those of Group 2, [Table 4](#tbl0025){ref-type="table"}. These differences became smaller during ISS03, to the point of no longer reaching statistical significance, except for TF and VLF spectral power, [Table 4](#tbl0025){ref-type="table"}. In other words, the two groups differed less in space (ISS03) than before flight.

Before flight, the BRAC amplitude was found to be much smaller in Group 1 as compared to Group 2, the difference being statistically significant for all considered HRV endpoints, except for LF, [Table 4](#tbl0025){ref-type="table"} (left). During ISS03, the 90-min amplitude increased in Group 1 and mostly decreased in Group 2 (except for LF), so that differences between the two groups were no longer statistically significant after spending several months in space, [Table 4](#tbl0025){ref-type="table"} (right). Similar results were observed for the 24-hour amplitude, and to a lesser extent for the 12-hour and 8-hour amplitudes of these HRV endpoints. These results suggest that the HRV of astronauts in Group 2, but not in Group 1, may have been sufficiently large to be exposed to the space environment.

4. Discussion {#sec0075}
=============

Spaceflight dramatically alters cardiovascular dynamics, as illustrated by changes in HRV [@bib0060] and a less negative slope β of the fractal scaling [@bib0045] confirmed herein. Kleitman\'s about 90-min BRAC [@bib0100] was found to be amplified about 3-fold in space, notably among astronauts of Group 1, in keeping with a corresponding increase in ULF01/TF (0.0001--0.0003 Hz, i.e., 55--166 min) and corresponding decreases in ULF02/TF and ULF03/TF. Major changes observed in space all relate to the same frequency range centered around one cycle in about 90 min, including β.

Beyond the partly built-in circadian rhythms [@bib0115], there are many other oscillations of different frequencies, including the BRAC, observed in the sleep-wake (REM/NREM) cycle and also in heart rate variability. Some neuropeptides can have more prominent ultradian (with a frequency higher than one cycle per day; e.g., 8-hour periodicity) than circadian changes [@bib0120]. We previously showed that the circadian rhythm persisted in space in HR and β \[[@bib0045], [@bib0060]\]. Herein, we confirm the presence in space of 24-, 12-, and 8-hour components in several HRV endpoints by the fit of a model including 4 anticipated components.

The question may be raised, however, whether different daily routines before and during flight (including higher or lower frequency of physical activities) as well as different sleep patterns in space may have contributed to the findings [@bib0125]. Whereas further work is needed to address this question, it should be noted that the space environment had a different effect on astronauts from the 2 groups. Amplitudes of all 4 anticipated components were markedly increased in astronauts of Group 1, whereas they were mostly decreased in astronauts of Group 2. It thus seems unlikely that the daily routine on the ISS fully accounts for the results observed in this study.

Unlike short-term (\<24 h) analysis of HRV \[[@bib0125], [@bib0130], [@bib0135]\], transient changes of body movement related to the daily routine were not associated with measurements of long HRV signals, including the ULF and VLF components and the slope β. Aoyagi et al. \[[@bib0050], [@bib0055]\] reported that during both usual daily-routine and constant-routine protocols in healthy men, HRV at frequencies between 0.0033 Hz and 10^−3.5^ Hz (25 sec to 57 min periods) was behavior-independent, possibly reflecting intrinsic mechanisms of the regulatory system. Amaral LAN et al. [@bib0140] also reported that the complexity of heartbeat dynamics showed behavioral-independent features during a constant-routine protocol. As reported previously \[[@bib0125], [@bib0130], [@bib0135]\], however, body movement was lower and the HF component of HRV was higher during sleep than during wakefulness. The less negative slope β in space versus Earth was also seen more prominently during the awake span [@bib0045]. Future studies are thus needed to examine how different daily routines before and during flight, including different sleep patterns in space, may contribute to our findings herein.

The presence of the BRAC in HRV endpoints observed herein is supported by different studies in a number of physiological systems. Based on 24-hour polygraphic tracings, Othmer E et al. [@bib0145] inferred that the so-called sleep-dream cycle of human sleep is a general activity pattern of the brain. Bailey D et al. [@bib0150] found regular oscillations with periods of 1--2 hours in their subjects\' oxygen consumption. Orr WC et al. [@bib0155] noted that their subjects\' heart rate showed the same about 90-min periodicity in performance of a complex vigilance task. Hiatt JF and Kripke DF [@bib0160] reported on 90- to 120-min ultradian rhythms in gastric motility. Lavie P and Kripke DF [@bib0165] discerned a cycle of 80--133 min in urine flow of awake subjects. The rhythm in urine flow was, however, clearly out of phase with those of electrolyte concentrations and osmolarity. Lavie P and Scherson A [@bib0170] observed rhythmic variations in subjects\' ability to fall asleep throughout the day. Conversely, an expected variation in vigilance was reported by Okawa M et al., [@bib0175]; the ultradian rhythms in vigilance had periods of 90--120 min.

The BRAC may play an important and unique role in keeping the quality of life in space independently of or in conjunction with the circadian rhythm. It is involved in the functioning of the central nervous system which integrates many somatic, visceral, and neurobehavioral functions and manifests itself in the alternation of non-REM and REM sleep. Ultradians may be the basic signature of life [@bib0180].

Effects of space weather are enormous, which have acted as selective forces in humans on Earth and shaped human life as we know it today. Using 61 worldwide populations, Hancock AM et al. [@bib0185] elucidated the genetic basis for adaptation to the climate-mediated selection in a scan of the human genome. They identified genes that are key to the differentiation of brown adipocytes, and genes whose regulation makes a difference in response to ultraviolet radiation [@bib0185]. Among the circadian clock components, cryptochrome may have played a pivotal role in evolution because it coordinates light-induced effects and protects from hazards of ultraviolet radiation [@bib0190]. Brown adipocytes and their cryptochromes may not only be relevant to survival and adaptation, but they may also be targeted by natural selection [@bib0195]. Circadian clocks in brown adipocytes are relevant to mammalian adaptation and the cryptochromes in particular are of key importance because of their evolutionary roots of circadian clocks.

Brown adipose tissue expressing BRAC may be an active pacemaker tissue, participating in the arrangement of ultradian [@bib0200] to infradian [@bib0205] oscillations. Circadian clocks may thus be built on properties generating metabolic oscillations in the ultradian range [@bib0190]. Brown adipose tissue may be a site of interaction between metabolic and circadian systems. A non-transcriptional pathway for the metabolic cycle engages the circadian clock, thereby enhancing clock performance [@bib0210]. As cryptochromes are key components of the core of the transcription-translation feedback loops on which circadian clocks are built, the question may thus be raised whether the amplification of the BRAC in space observed herein is a sign of early adaptation to microgravity.

5. Conclusion {#sec0080}
=============

Whether the increase in space of the BRAC amplitude is a sign that the intrinsic autonomic regulatory system may start to adapt requires further investigation, as β remains disturbed throughout the 6-month spaceflight. Whether some features of the HRV may indicate suitability for space travel also deserves further work as the BRAC amplification in space was only observed in some but not all astronauts. Most HRV changes observed in space relate to a frequency window centered around one cycle in about 90 min, although astronauts follow regular 24-hour rest-activity and feeding schedules on the ISS. Since the BRAC component is amplified in space for only specific HRV endpoints, it is likely to represent a physiologic response rather than an artifact from the ISS orbit. If so, it may offer a way to help adaptation to microgravity during long-duration spaceflight.

Declarations {#sec0085}
============

Author contribution statement {#sec0090}
-----------------------------

Kuniaki Otsuka: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Germaine Cornelissen, Yutaka Kubo, Mitsutoshi Hayashi, Koichi Shibata, Koh Mizuno: Analyzed and interpreted the data; Wrote the paper.

Satoshi Furukawa, Tatsuya Aiba, Hiroshi Ohshima, Chiaki Mukai: Conceived and designed the experiments; Performed the experiments; Wrote the paper.

Competing interest statement {#sec0095}
----------------------------

The authors declare no conflict of interest.

Additional information {#sec0100}
----------------------

No additional information is available for this paper.

Funding statement {#sec0105}
-----------------

The JAXA Chronobiology Project was supported by the Japan Aerospace Exploration Agency (KO, YK, MH, NY, KS, TA, SF, HO, CM), Halberg Chronobiology Fund (GC).

The authors thank Dr. I. Tayama and S. Ishida from the Space Biomedical Research Group, Japan Aerospace Exploration Agency (JAXA), for cooperation in our study. The authors also acknowledge the cooperation of the astronauts, the engineers, staff and managers of JAXA and NASA.

![Illustrative examples of the 4-component model fitted to the TF spectral power of two astronauts during a 6-month spaceflight. As compared to preflight (left), the 90-min component is amplified during session ISS03 in space (right). Whereas the circadian amplitude is mostly unchanged in one case ([Fig. 1](#fig0005){ref-type="fig"}A), it is also amplified in another case ([Fig. 1](#fig0005){ref-type="fig"}B). A fixed model is used, considering only anticipated periodicities. As such, the model is not optimal for any given record, even if on a group basis it conveys the behavior of components that are the most commonly detected in such records. Because it is a fixed model, the residual variance may exhibit lack of fit. Nevertheless, the amplitude of the about 90-min component is increased during ISS03 in astronauts of Group 1, when it resembles that of astronauts of Group 2.](gr1){#fig0005}

###### 

Change in characteristics of heart rate variability associated with 6-month mission in space: Numerical results.^\*^

Table 1A

  Variable                                Units                     Target period (range)   n        Control (Before flight)   ISS01    ISS02    ISS03    After flight                                       
  ---------------------------- ---------- ------------------------- ----------------------- -------- ------------------------- -------- -------- -------- -------------- -------- -------- -------- -------- -------
  Time- domain measures        HR         (beats/min)               24 hours                10       69.9                      10.9     66.7     8.5      66.9           7.0      66.6     7.4      69.2     8.9
  NN-interval                  (msec)     24 hours                  10                      878.2    146.7                     914.1    126.4    906.4    97.6           911.9    104.3    880.5    120.9    
  SDNN                         (msec)     24 hours                  10                      132.5    45.2                      148.4    29.5     140.1    52.6           151.0    43.2     144.7    43.5     
  SDANN (5 min)                (msec)     24 hours                  10                      115.8    43.6                      129.0    27.0     121.4    46.0           130.0    39.3     125.1    43.2     
  SDANN (30 min)               (msec)     24 hours                  10                      109.3    44.2                      125.2    27.1     117.9    44.7           129.1    38.5     116.8    44.6     
  TINN                         (msec)     24 hours                  10                      571.5    178.9                     638.0    144.9    523.5    186.7          552.7    128.7    612.3    146.9    
  HRVI                         (--)       24 hours                  10                      35.7     11.2                      39.9     9.1      32.7     11.7           34.5     8.0      38.3     9.2      
  Triangular Index (TI)        (--)       24 hours                  10                      34.2     10.4                      38.3     9.2      30.8     10.8           31.8     7.2      36.8     9.0      
  Lorenz Plot Length           (msec)     24 hours                  10                      627.9    228.3                     690.7    160.7    659.0    284.5          745.0    252.2    707.1    234.9    
  Lorenz Plot Width            (msec)     24 hours                  10                      54.9     16.5                      50.9     15.6     51.5     13.7           61.7     15.9     58.9     15.8     
  Length/Width ratio           (--)       24 hours                  10                      11.5     2.5                       14.5     4.2      13.0     4.9            12.5     4.2      12.5     4.8      
  SDNN index (30 min)          (msec)     30 min                    10                      72.3     19.1                      66.6     16.7     63.9     15.1           68.0     17.6     76.6     17.4     
  SDNN index (5 min)           (msec)     5 min                     10                      56.5     14.8                      53.1     13.2     50.9     11.3           55.1     13.4     58.8     13.4     
  CVNN                         (%)        5 min                     10                      16.3     5.1                       17.5     4.9      16.0     4.6            17.3     3.6      17.7     5.9      
  r-MSSD                       (msec)     5 min                     10                      23.9     5.9                       23.1     5.9      22.6     5.2            26.4     5.8      24.7     6.6      
  NN50                         (number)   5 min                     10                      4048.2   2841.3                    3603.4   2514.5   3142.4   2605.1         4442.1   2610.0   4226.3   2616.1   
  pNN50                        (%)        5 min                     10                      4.360    3.536                     4.050    3.143    3.430    2.819          5.820    3.594    5.090    4.260    
  Frequency- domain measures   \|β\|      (log(msec^2^)/log(Hz))    90 min (1.7--166 min)   10       1.087                     0.130    0.977    0.098    0.910          0.130    0.924    0.095    1.135    0.147
  TF-component                 msec^2^    90 min (2 sec--166 min)   10                      6417.1   3238.0                    5932.1   2453.4   5297.5   2806.2         6530.9   3562.3   6897.6   2823.3   
  ULF-component                msec^2^    90 min (5--166 min)       10                      3479.8   1636.4                    3255.5   1295.1   2857.4   1982.5         3624.3   2362.4   3815.4   1605.2   
  ULF01                        msec^2^    90 min (55--166 min)      10                      1361.2   775.7                     1788.0   747.4    1450.9   1146.7         2080.8   1399.7   1389.3   640.7    
  ULF02                        msec^2^    36 min (17--55 min)       10                      1190.3   561.6                     885.2    433.2    849.6    561.5          878.2    520.4    1378.1   548.7    
  ULF03                        msec^2^    10 min (3--17 min)        10                      1360.3   596.7                     920.2    522.1    868.0    488.2          1034.7   764.3    1533.5   860.7    
  VLF-component                msec^2^    5 min (25 sec--5 min)     10                      2113.7   1361.6                    1928.7   1034.7   1741.5   827.4          2105.8   1211.2   2210.5   1127.5   
  VLF01                        msec^2^    2 min (50 sec-3.3 min)    10                      1177.2   834.2                     1114.4   605.5    1002.5   464.8          1245.3   758.5    1209.6   666.5    
  VLF02                        msec^2^    42 sec (33--50 sec)       10                      291.9    185.9                     275.2    151.7    250.3    132.3          287.6    147.5    297.8    134.0    
  VLF03                        msec^2^    20 sec (6.7--33 sec)      10                      911.3    425.2                     836.9    416.2    773.5    367.4          864.6    389.8    960.4    391.9    
  LF-component                 msec^2^    15 sec (6--25 sec)        10                      698.8    316.1                     635.8    329.3    595.8    296.1          661.1    306.0    742.9    310.6    
  HF-component                 msec^2^    4.3 sec (2.5--6 sec)      10                      116.5    55.1                      104.6    60.2     94.9     45.9           127.6    63.7     120.1    52.2     
  LF/HF ratio                  (--)                                 10                      6.428    2.711                     6.398    1.760    6.305    0.744          5.606    1.761    6.506    2.129    
  ULF/TF                       (--)       90 min (5--166 min)       10                      0.549    0.079                     0.556    0.071    0.511    0.123          0.542    0.091    0.557    0.092    
  ULF01/TF                     (--)       90 min (55--166 min)      10                      0.207    0.053                     0.314    0.078    0.251    0.095          0.310    0.090    0.202    0.070    
  ULF02/TF                     (--)       36 min (17--55 min)       10                      0.189    0.037                     0.145    0.025    0.154    0.051          0.136    0.030    0.204    0.045    
  ULF03/TF                     (--)       10 min (3--17 min)        10                      0.219    0.035                     0.151    0.034    0.164    0.024          0.151    0.034    0.219    0.047    
  VLF-/TF                      (--)       5 min (25 sec--5 min)     10                      0.316    0.057                     0.319    0.064    0.347    0.088          0.323    0.055    0.312    0.065    
  VLF01/TF                     (--)       2 min (50 sec-3.3 min)    10                      0.173    0.041                     0.186    0.043    0.200    0.050          0.189    0.039    0.169    0.042    
  VLF02/TF                     (--)       42 sec (33--50 sec)       10                      0.044    0.010                     0.045    0.013    0.051    0.021          0.045    0.011    0.043    0.013    
  VLF03/TF                     (--)       20 sec (6.7--33 sec)      10                      0.147    0.045                     0.139    0.038    0.159    0.055          0.143    0.055    0.143    0.047    
  LF-/TF                       (--)       15 sec (6--25 sec)        10                      0.114    0.039                     0.106    0.034    0.122    0.044          0.110    0.047    0.111    0.040    
  HF-/TF                       (--)       4.3 sec (2.5--6 sec)      10                      0.019    0.009                     0.018    0.007    0.020    0.008          0.023    0.014    0.018    0.008    

^\*^For definition of HRV endpoints, see [@bib0075].

###### 

Comparison of relative HRV endpoints in Space and on Earth.[\*](#tblfn0005){ref-type="table-fn"}

Table 1B

              *Means (10 astronauts)*                     Space vs. Earth   ISS01-03                                                
  ----------- ------------------------- -------- -------- ----------------- ---------- -------- -------- ------- ---------- ------- -------
              Primary endpoints                                                                                                     
  ULF01       83.36                     121.19   81.97    124.52            88.95      86.16    109.23   1.933   NS         3.106   NS
  ULF02       115.85                    85.24    76.54    85.12             137.25     126.55   82.30    6.265   0.001      0.431   NS
  ULF03       123.56                    80.66    75.23    84.86             135.70     129.63   80.25    7.344   \< 0.001   0.924   NS
  VLF01       97.16                     99.97    91.02    107.01            104.83     101.00   99.34    0.250   NS         2.135   NS
  VLF02       100.69                    97.34    90.57    103.60            107.80     104.24   97.17    1.354   NS         2.141   NS
  VLF03       104.67                    94.54    90.20    99.38             111.21     107.94   94.71    2.345   NS         1.327   NS
  LF          105.48                    93.15    90.01    98.99             112.37     108.92   94.05    2.160   NS         1.153   NS
  HF          103.99                    90.11    85.09    112.54            108.27     106.13   95.91    1.121   NS         4.582   NS
                                                                                                                                    
              Secondary endpoints                                                                                                   
  TF          102.32                    97.44    83.44    103.19            113.61     107.96   94.69    2.482   NS         3.778   NS
  ULF         102.95                    99.96    78.42    103.52            115.15     109.05   93.97    1.910   NS         2.621   NS
  VLF         100.96                    96.99    88.87    103.20            109.98     105.47   96.36    1.630   NS         2.321   NS
  ULF/TF      101.16                    103.07   93.37    99.96             102.44     101.80   98.80    0.906   NS         1.214   NS
   ULF01/TF   81.13                     124.29   96.43    119.89            78.25      79.69    113.54   4.376   0.014      2.416   NS
   ULF02/TF   114.49                    88.38    91.39    82.87             122.87     118.68   87.55    6.199   0.001      0.562   NS
   ULF03/TF   121.54                    83.79    91.27    83.26             120.15     120.84   86.11    6.945   0.001      1.100   NS
  VLF/TF      97.60                     99.31    107.00   100.06            96.03      96.81    102.12   1.145   NS         0.555   NS
   VLF01/TF   93.69                     101.89   109.48   103.35            91.59      92.64    104.91   2.137   NS         0.397   NS
   VLF02/TF   96.91                     100.07   108.81   100.47            93.74      95.32    103.12   1.530   NS         0.621   NS
   VLF03/TF   101.92                    96.84    107.65   97.09             96.50      99.21    100.53   0.175   NS         1.169   NS
  LF/TF       102.99                    95.35    107.26   96.97             97.42      100.21   99.86    0.038   NS         1.189   NS
  HF/TF       101.09                    92.76    102.21   109.37            94.58      97.83    101.45   0.436   NS         1.312   NS
  LF/HF       100.55                    102.16   103.28   89.54             104.47     102.51   98.33    0.484   NS         1.974   NS
  HR          102.68                    98.28    98.82    98.22             102.00     102.34   98.44    1.793   NS         0.043   NS
  NN          97.40                     101.77   101.19   101.64            98.00      97.70    101.53   1.788   NS         0.035   NS
  CVRR        94.98                     103.46   94.89    102.43            104.23     99.61    100.26   0.119   NS         0.613   NS
  SDNN        91.44                     106.14   96.17    105.38            100.86     96.15    102.57   1.139   NS         1.053   NS
  r-MSSD      98.74                     95.33    93.78    110.11            102.04     100.39   99.74    0.161   NS         4.545   NS
  NN          105.87                    100.09   97.18    96.38             100.48     103.17   97.88    1.544   NS         0.698   NS
  NN50        104.53                    92.78    79.83    113.64            110.59     107.56   94.28    1.093   NS         1.980   NS
  NN50+       96.13                     92.98    78.64    126.63            105.63     100.88   99.42    0.113   NS         2.541   NS
  NN50-       95.24                     82.15    75.63    137.51            109.47     102.36   98.43    0.257   NS         4.117   NS
  pNN50       90.37                     87.90    77.29    135.50            108.94     99.65    100.23   0.035   NS         3.388   NS
  pNN50+      87.99                     93.91    79.83    130.65            107.61     97.80    101.46   0.233   NS         2.578   NS
  pNN50−      90.21                     86.15    73.10    140.81            109.74     99.97    100.02   0.002   NS         4.296   NS
  SDANN5      92.10                     107.08   95.96    104.85            100.01     96.05    102.63   1.046   NS         1.187   NS
  SDANN30     90.01                     107.93   96.77    108.65            96.63      93.32    104.45   1.537   NS         1.339   NS
  SDmean5     102.51                    96.81    93.19    100.27            107.22     104.87   96.76    4.004   0.025      3.693   NS
  SDmean30    103.63                    95.80    92.12    97.68             110.77     107.20   95.20    6.551   0.001      1.954   NS
  N           94.98                     99.95    106.92   107.43            90.72      92.85    104.77   2.359   NS         1.181   NS
  X           98.29                     100.10   100.54   98.89             102.18     100.24   99.84    0.184   NS         0.237   NS
  M           96.69                     105.11   98.51    101.23            98.46      97.58    101.61   2.598   NS         3.447   NS
  TINN        97.65                     111.12   88.84    95.94             106.45     102.05   98.63    0.898   NS         6.227   0.048
  HRVI        97.64                     111.14   88.83    95.95             106.44     102.04   98.64    0.891   NS         6.241   0.047
  TI          98.60                     112.06   88.28    93.14             107.92     103.26   97.83    1.374   NS         7.098   0.027
  Length      90.65                     103.84   94.15    108.23            103.14     96.89    102.07   0.823   NS         1.231   NS
  Width       97.90                     91.20    92.98    111.42            106.50     102.20   98.53    0.766   NS         4.756   NS
  Len/Wid     91.94                     113.80   101.01   96.52             96.73      94.34    103.78   1.606   NS         1.371   NS
                                                                                                                                    
  Trend (β)   108.03                    97.23    90.33    91.80             112.61     110.32   93.12    4.298   0.016      1.958   NS

P-values adjusted for multiple testing, using Bonferroni\'s inequality, considering that 8 different tests were conducted (in 8 independent frequency regions).

Secondary endpoints also used the same correcting factor, considering the large correlation among different endpoints, shown here for sake of completeness only (rather than for testing per se).

For definition of HRV endpoints, see [@bib0075].

24-hour mean HRV endpoints expressed as a percentage of 5-session average for each astronaut, then averaged during each session across the 10 astronauts.

###### 

Individual HRV responses of astronauts.[\*](#tblfn0010){ref-type="table-fn"}

Table 2

  Subjects       Variables    units        Control (Before flight)   ISS01   ISS02   ISS03   After flight                                          
  -------------- ------------ ------------ ------------------------- ------- ------- ------- -------------- ------- ------- ------- ------- ------ ------
  Group 1        Case 1       Heart Rate   (b/min)                   74.8    10.6    81.5    16.1           75.5    7.3     71.6    12.2    73.5   16.9
  r-MSSD         (msec)       23.1         8.2                       22.6    5.7     23.3    5.3            27.0    7.1     28.9    9.3            
  pNN50          (%)          3.9          5.9                       3.2     2.8     3.4     2.5            5.4     3.8     8.0     6.2            
  HF-component   msec^2^      127.5        112.9                     92.1    54.3    100.4   49.7           127.5   61.1    193.1   103.4          
  LF/HF ratio    (--)         6.0          3.4                       6.6     3.0     7.1     2.7            7.3     3.7     4.9     2.5            
  Case 2         Heart Rate   (b/min)      78.8                      13.5    67.0    9.2     77.7           9.8     74.8    7.1     78.1    10.0   
  r-MSSD         (msec)       23.3         7.6                       29.5    6.7     23.6    6.3            26.1    6.6     23.8    7.4            
  pNN50          (%)          4.5          4.8                       8.4     5.2     4.4     3.8            6.0     5.9     4.8     4.7            
  HF-component   msec^2^      169.1        114.7                     235.0   106.9   162.5   101.9          239.4   170.0   165.1   123.1          
  LF/HF ratio    (--)         6.0          4.1                       6.0     3.0     6.5     3.5            5.4     3.1     6.3     4.4            
  Case 3         Heart Rate   (b/min)      89.9                      11.9    72.2    14.4    70.9           8.0     78.5    12.5    82.3    7.5    
  r-MSSD         (msec)       22.9         3.9                       20.3    6.6     18.2    4.5            32.9    13.7    19.2    4.7            
  pNN50          (%)          3.0          2.5                       2.4     3.7     1.4     2.2            13.1    13.8    1.5     2.0            
  HF-component   msec^2^      105.3        48.0                      86.0    49.3    71.3    34.3           180.1   153.0   98.0    64.0           
  LF/HF ratio    (--)         5.8          2.8                       5.8     3.6     6.2     3.8            3.3     2.6     6.2     3.2            
  Case 4         Heart Rate   (b/min)      77.3                      10.0    70.3    15.0    64.9           6.2     66.4    14.6    66.9    6.1    
  r-MSSD         (msec)       16.8         4.9                       17.8    3.9     19.7    3.7            20.2    4.4     23.0    6.2            
  pNN50          (%)          1.3          1.9                       1.2     1.4     1.5     1.6            1.9     1.8     3.8     3.8            
  HF-component   msec^2^      55.7         33.2                      56.3    27.7    74.0    30.1           69.2    29.3    102.7   66.9           
  LF/HF ratio    (--)         13.4         7.2                       10.7    6.1     7.4     3.2            9.2     5.5     8.1     4.6            
  Case 5         Heart Rate   (b/min)      61.6                      5.1     59.9    6.7     56.5           9.0     57.2    6.7     62.1    10.6   
  r-MSSD         (msec)       15.9         2.9                       11.9    2.2     15.1    3.7            15.2    3.6     13.1    3.8            
  pNN50          (%)          0.5          0.6                       0.2     0.4     0.7     1.2            0.6     1.5     0.3     0.7            
  HF-component   msec^2^      37.9         15.7                      22.0    10.3    31.5    15.3           31.8    17.1    26.2    19.2           
  LF/HF ratio    (--)         4.6          2.7                       7.5     4.6     7.5     4.6            6.6     4.7     7.4     4.8            
  Case 6         Heart Rate   (b/min)      69.0                      7.4     62.4    6.7     67.7           12.9    63.4    12.4    71.7    12.0   
  r-MSSD         (msec)       19.0         4.4                       22.3    7.1     20.4    5.7            31.1    8.5     23.4    5.6            
  pNN50          (%)          1.8          1.8                       3.5     4.1     2.3     2.6            10.2    6.8     4.0     3.6            
  HF             msec^2^      69.1         38.6                      90.6    54.7    84.7    62.6           144.5   88.9    110.2   55.0           
  LF/HF ratio    (--)         10.8         6.4                       8.7     5.4     8.9     5.2            5.7     3.3     12.2    7.3            
  Case 7         Heart Rate   (b/min)      64.6                      5.4     66.2    10.4    64.5           5.9     62.9    5.6     66.6    6.4    
  r-MSSD         (msec)       21.3         3.8                       25.2    7.1     18.1    5.5            22.6    4.6     18.8    3.5            
  pNN50          (%)          2.2          1.8                       5.7     5.4     1.5     2.1            3.2     3.1     1.4     1.3            
  HF-component   msec^2^      79.1         32.9                      85.6    42.3    50.4    25.2           78.2    30.6    62.6    25.5           
  LF/HF ratio    (--)         5.9          3.8                       4.7     3.0     6.1     3.7            4.8     2.7     7.5     4.7            
  Group 2        Case 8       Heart Rate   (b/min)                   65.8    13.5    65.5    11.2           69.4    15.8    67.7    14.4    64.5   7.2
  r-MSSD         (msec)       28.8         9.7                       21.0    4.8     28.1    4.7            25.2    6.3     36.8    6.0            
  pNN50          (%)          8.4          9.1                       2.4     2.4     6.5     3.7            4.8     4.8     15.3    6.2            
  HF-component   msec^2^      160.9        129.4                     73.4    39.9    91.7    39.5           90.7    48.2    163.1   65.9           
  LF/HF ratio    (--)         8.6          6.9                       10.3    7.3     6.7     3.3            8.4     4.9     5.9     2.7            
  Case 9         Heart Rate   (b/min)      67.8                      16.5    71.8    19.0    65.2           10.5    66.4    13.0    76.6    23.0   
  r-MSSD         (msec)       33.6         12.5                      27.6    8.4     32.9    8.0            32.7    10.6    27.7    10.3           
  pNN50          (%)          12.2         11.3                      7.0     6.4     11.1    8.0            10.9    9.7     7.3     8.8            
  HF-component   msec^2^      200.7        137.5                     147.7   80.0    179.8   92.5           201.3   135.5   123.1   90.0           
  LF/HF ratio    (--)         7.8          4.2                       7.5     3.7     7.2     3.1            6.3     2.7     8.9     4.5            
  Case 10        Heart Rate   (b/min)      51.6                      8.5     49.8    7.0     57.1           17.9    54.4    13.5    53.1    7.4    
  r-MSSD         (msec)       35.3         8.8                       32.9    6.5     26.5    6.3            31.6    7.7     31.7    6.6            
  pNN50          (%)          13.2         8.8                       10.9    6.4     5.0     4.3            9.7     6.7     9.7     6.2            
  HF-component   msec^2^      161.7        78.7                      157.7   55.6    102.4   42.6           119.9   45.2    139.8   60.2           
  LF/HF ratio    (--)         6.7          4.7                       5.7     3.6     6.0     3.6            6.3     3.6     6.9     4.7            

r-MSSD: square root of mean squared differences of successive NN intervals; pNN50: fraction of consecutive NN intervals that differ by more than 50 ms; HF-component: spectral power centered around 3.6 sec; LF/HF ratio: ratio of low-frequency (LF, centered around 10.5 sec) and high-frequency (HF) spectral power; all indices obtained from 5-min segments, averaged over the entire 24-hour span.

Astronauts were grouped in terms of their NN records (see text). Each record contains 254 to 286 values, except for case 8 after return to Earth (N = 70 or 71).

###### 

Change in relative amplitude of 24-, 12-, 8-, and 1.5-hour components of some HRV endpoints during 6-month mission in space.[\*](#tblfn0015){ref-type="table-fn"}

Table 3

             Group 1 (N = 7)                                                                                    
  ---------- ----------------- -------- ------- -------- ------- ------- -------- ------- ------------- ------- -----------
  NN                                                                                                            
   24h-A     8.07              12.52    9.80    12.51    9.85    8.96    11.61    1.940   NS            2.332   NS
   12h-A     5.14              5.95     6.26    6.70     6.70    5.92    6.30     0.399   NS            1.749   NS
   8h-A      3.74              4.42     3.03    4.31     3.66    3.70    3.92     0.219   NS            0.831   NS
   1.5h-A    0.94              1.16     0.99    1.61     1.42    1.18    1.25     0.294   NS            1.977   NS
  β                                                                                                             
   24h-A     18.35             20.67    20.86   20.48    19.60   18.98   20.67    0.275   NS            0.250   NS
   12h-A     13.31             19.97    20.67   19.06    12.45   12.88   19.90    1.195   NS            0.843   NS
   8h-A      13.67             13.35    12.31   12.07    12.25   12.96   12.58    0.380   NS            0.581   NS
   1.5h-A    2.07              1.68     1.76    1.99     1.33    1.70    1.81     0.326   NS            0.222   NS
  TF                                                                                                            
   24h-A     24.59             56.39    53.79   62.60    33.99   29.29   57.59    3.978   **0.044**     2.326   NS
   12h-A     23.94             43.43    44.11   49.68    28.03   25.98   45.74    2.531   NS            1.916   NS
   8h-A      18.99             38.89    39.86   41.00    19.76   19.38   39.92    5.149   **0.013**     2.877   0.169
   1.5h-A    3.17              8.14     7.76    11.29    4.93    4.05    9.06     3.367   ***0.091***   3.240   0.106
  ULF                                                                                                           
   24h-A     37.33             97.94    72.79   104.14   49.16   43.25   91.62    3.686   ***0.062***   2.382   NS
   12h-A     33.64             84.49    66.30   88.82    37.76   35.70   79.87    3.417   ***0.085***   2.644   NS
   8h-A      30.91             63.26    54.71   60.19    32.81   31.86   59.39    3.484   ***0.078***   2.278   NS
   1.5h-A    4.06              11.14    8.47    15.80    5.71    4.88    11.80    7.485   **0.002**     4.923   **0.016**
  ULF01                                                                                                         
   24h-A     42.91             158.90   95.59   166.67   43.62   43.26   140.39   4.465   **0.026**     2.601   NS
   12h-A     41.33             144.99   92.88   145.30   43.58   42.46   127.72   3.302   ***0.098***   2.188   NS
   8h-A      39.90             110.80   74.42   105.45   40.78   40.34   96.89    3.014   0.141         2.145   NS
   1.5h-A    8.87              22.71    16.30   31.32    11.55   10.21   23.44    4.706   **0.020**     4.052   **0.040**
  ULF02                                                                                                         
   24h-A     44.92             61.12    70.78   51.16    71.49   58.20   61.02    0.549   NS            0.742   NS
   12h-A     44.73             55.49    58.22   48.84    49.83   47.28   54.18    0.778   NS            0.426   NS
   8h-A      34.01             32.05    47.92   35.97    51.07   42.54   38.65    0.655   NS            0.183   NS
   1.5h-A    4.44              4.71     4.79    6.13     4.76    4.60    5.21     0.917   NS            1.647   NS
  ULF03                                                                                                         
   24h-A     62.70             36.69    40.41   49.54    67.57   65.13   42.22    2.574   NS            0.772   NS
   12h-A     41.72             32.60    26.97   28.96    34.97   38.35   29.51    1.480   NS            1.653   NS
   8h-A      29.02             19.52    26.48   33.48    26.66   27.84   26.49    0.178   NS            0.529   NS
   1.5h-A    3.01              2.01     2.38    3.01     2.50    2.75    2.47     0.621   NS            0.008   NS
  ULF/TF                                                                                                        
   24h-A     17.86             28.05    20.27   18.86    14.31   16.09   22.39    1.938   NS            0.228   NS
   12h-A     16.58             25.73    20.24   22.24    15.18   15.88   22.74    1.659   NS            1.168   NS
   8h-A      16.19             15.74    15.62   13.76    11.14   13.67   15.04    0.514   NS            0.557   NS
   1.5h-A    3.97              4.83     4.87    5.28     3.75    3.86    4.99     1.753   NS            0.869   NS
  ULF01/TF                                                                                                      
   24h-A     45.30             68.04    31.24   71.25    88.99   67.14   56.84    0.507   NS            1.408   NS
   12h-A     32.19             67.20    41.99   59.94    27.71   29.95   56.38    2.437   NS            1.721   NS
   8h-A      34.96             41.44    30.34   47.92    29.27   32.11   39.90    0.950   NS            1.299   NS
   1.5h-A    10.25             10.52    11.04   14.97    7.09    8.67    12.18    2.771   NS            1.772   NS
  ULF02/TF                                                                                                      
   24h-A     35.43             29.49    33.98   18.14    30.42   32.93   27.20    1.266   NS            3.291   0.100
   12h-A     33.34             30.88    26.52   28.13    27.31   30.32   28.51    0.319   NS            0.646   NS
   8h-A      15.88             15.95    18.20   27.22    33.30   24.59   20.45    0.963   NS            1.769   NS
   1.5h-A    6.22              6.01     5.94    6.98     7.83    7.02    6.31     0.658   NS            0.424   NS
  ULF03/TF                                                                                                      
   24h-A     53.53             24.10    26.26   41.43    39.95   46.74   30.60    3.376   **0.090**     1.266   NS
   12h-A     26.80             22.56    13.09   16.51    18.06   22.43   17.39    1.215   NS            1.599   NS
   8h-A      16.01             13.82    14.19   17.23    23.01   19.51   15.08    1.052   NS            0.363   NS
   1.5h-A    5.33              4.07     5.23    6.62     4.51    4.92    5.31     0.491   NS            1.837   NS
  VLF                                                                                                           
   24h-A     29.89             41.13    38.29   42.63    34.64   32.27   40.68    0.859   NS            1.013   NS
   12h-A     20.71             24.11    20.84   29.59    21.58   21.15   24.85    0.964   NS            1.102   NS
   8h-A      17.50             26.51    25.08   33.56    10.67   14.08   28.38    2.857   NS            1.621   NS
   1.5h-A    9.68              17.06    14.18   17.14    11.36   10.52   16.13    1.717   NS            1.436   NS
  LF                                                                                                            
   24h-A     18.12             15.07    18.21   24.30    27.81   22.97   19.19    1.267   NS            0.760   NS
   12h-A     14.30             18.91    13.14   17.69    24.28   19.29   16.58    0.729   NS            0.639   NS
   8h-A      19.79             14.11    11.82   17.08    22.48   21.14   14.34    1.485   NS            0.524   NS
   1.5h-A    7.24              7.96     5.75    8.92     5.88    6.56    7.54     0.802   NS            0.952   NS
  HF                                                                                                            
   24h-A     26.30             27.31    26.59   55.76    36.39   31.35   36.56    0.482   NS            1.266   NS
   12h-A     18.17             17.89    20.99   38.32    30.69   24.43   25.73    0.152   NS            1.794   NS
   8h-A      16.34             17.95    16.92   25.72    18.22   17.28   20.20    0.635   NS            1.502   NS
   1.5h-A    6.79              6.77     6.85    10.87    12.54   9.67    8.16     0.647   NS            1.358   NS
  LF/HF                                                                                                         
   24h-A     24.67             24.76    14.48   26.18    23.65   24.16   21.81    0.564   NS            0.289   NS
   12h-A     11.93             15.23    10.84   15.63    12.33   12.13   13.90    0.596   NS            1.087   NS
   8h-A      10.44             13.20    13.98   10.21    8.82    9.63    12.46    1.959   NS            0.054   NS
   1.5h-A    9.06              11.16    5.31    11.39    11.76   10.41   9.29     1.775   NS            0.875   NS

             Group 2 (N = 3)                                                                                   
  ---------- ----------------- ------- ------- -------- ------- ------- ------- -------- ------------- ------- -------------
  NN                                                                                                           
   24h-A     19.82             15.86   17.48   18.32    17.99   18.91   17.22   0.621    NS            0.499   NS
   12h-A     5.80              8.78    9.45    8.40     8.80    7.30    8.88    1.113    NS            0.777   NS
   8h-A      3.61              4.28    4.35    3.83     4.14    3.88    4.15    0.271    NS            0.129   NS
   1.5h-A    1.06              1.62    1.90    2.40     1.90    1.48    1.97    1.907    NS            2.546   NS
  β                                                                                                            
   24h-A     37.55             24.94   30.95   35.59    31.28   34.42   30.49   0.406    NS            0.210   NS
   12h-A     23.78             29.58   30.72   27.21    17.56   20.67   29.17   1.394    NS            0.486   NS
   8h-A      19.68             17.39   15.66   16.70    18.84   19.26   16.58   0.455    NS            1.043   NS
   1.5h-A    2.24              2.11    1.86    1.67     1.75    1.99    1.88    0.317    NS            0.557   NS
  TF                                                                                                           
   24h-A     60.22             31.50   51.61   39.07    35.60   47.91   40.73   0.760    NS            1.368   NS
   12h-A     48.94             17.76   45.86   50.37    33.90   41.42   38.00   0.338    NS            0.043   NS
   8h-A      47.92             26.41   35.01   33.84    18.12   33.02   31.75   0.916    NS            7.198   **0.002**
   1.5h-A    9.80              3.28    5.68    7.49     4.73    7.27    5.48    2.802    0.186         0.732   NS
  ULF                                                                                                          
   24h-A     87.09             53.23   73.05   58.51    45.77   66.43   61.60   0.283    NS            0.861   NS
   12h-A     75.45             44.29   65.30   71.00    38.67   57.06   60.20   0.238    NS            0.128   NS
   8h-A      76.57             46.30   55.71   63.55    35.12   55.85   55.19   0.085    NS            2.082   NS
   1.5h-A    13.05             8.83    8.23    5.60     6.02    9.54    7.55    1.275    NS            3.629   ***0.066***
  ULF01                                                                                                        
   24h-A     108.64            71.78   88.03   96.08    32.58   70.61   85.30   0.967    NS            0.375   NS
   12h-A     120.47            64.16   92.57   121.20   57.76   89.12   92.64   0.269    NS            0.050   NS
   8h-A      114.42            72.49   91.25   107.47   59.20   86.81   90.40   0.182    NS            0.339   NS
   1.5h-A    30.75             17.55   16.54   12.27    12.29   21.52   15.45   2.639    NS            6.623   **0.003**
  ULF02                                                                                                        
   24h-A     115.88            41.77   77.19   41.78    79.06   97.47   53.58   3.810    ***0.053***   4.063   **0.040**
   12h-A     103.51            39.62   54.65   41.31    47.27   75.39   45.19   4.227    **0.033**     3.225   0.108
   8h-A      64.38             33.04   41.18   37.45    36.76   50.57   37.22   2.777    NS            2.236   NS
   1.5h-A    10.37             5.16    7.12    3.82     5.24    7.81    5.37    1.047    NS            2.217   NS
  ULF03                                                                                                        
   24h-A     65.84             32.72   48.60   36.56    44.94   55.39   39.29   0.675    NS            0.810   NS
   12h-A     79.34             15.71   44.70   43.96    26.36   52.85   34.79   1.003    NS            0.886   NS
   8h-A      55.65             21.07   21.60   35.66    26.79   41.22   26.11   1.023    NS            0.677   NS
   1.5h-A    2.73              3.12    1.96    2.71     2.02    2.37    2.60    0.483    NS            0.008   NS
  ULF/TF                                                                                                       
   24h-A     24.97             22.74   29.37   31.76    29.00   26.99   27.96   0.107    NS            0.958   NS
   12h-A     23.48             26.92   26.49   26.60    15.56   19.52   26.67   3.320    ***0.096***   0.328   NS
   8h-A      23.18             20.18   17.73   17.54    19.29   21.23   18.48   0.726    NS            1.441   NS
   1.5h-A    3.93              4.16    8.16    5.99     6.37    5.15    6.11    0.615    NS            0.676   NS
  ULF01/TF                                                                                                     
   24h-A     42.32             44.30   48.37   50.48    31.22   36.77   47.72   0.776    NS            1.437   NS
   12h-A     47.50             46.01   42.67   58.27    39.76   43.63   48.98   0.904    NS            1.354   NS
   8h-A      50.49             55.09   50.52   49.11    49.18   49.83   51.57   0.267    NS            0.354   NS
   1.5h-A    9.85              11.57   18.14   11.70    9.94    9.90    13.81   1.159    NS            0.450   NS
  ULF02/TF                                                                                                     
   24h-A     30.10             29.26   41.42   27.29    54.72   42.41   32.66   1.034    NS            0.825   NS
   12h-A     56.17             26.10   26.97   25.47    35.02   45.60   26.18   2.146    NS            2.269   NS
   8h-A      22.72             13.42   10.26   17.50    42.38   32.55   13.73   13.648   **\<0.001**   1.159   NS
   1.5h-A    8.32              4.50    5.87    7.41     5.46    6.89    5.93    0.276    NS            0.167   NS
  ULF03/TF                                                                                                     
   24h-A     28.57             13.69   19.92   8.49     36.54   32.55   14.03   2.902    NS            2.763   NS
   12h-A     24.10             13.50   16.68   19.87    24.19   24.14   16.69   1.471    NS            0.489   NS
   8h-A      16.60             21.78   19.32   18.71    16.52   16.56   19.94   1.459    NS            0.378   NS
   1.5h-A    8.47              3.22    5.00    8.68     5.01    6.74    5.63    1.081    NS            0.192   NS
  VLF                                                                                                          
   24h-A     67.35             36.26   43.68   58.98    44.16   55.76   46.31   0.654    NS            0.982   NS
   12h-A     43.61             25.58   22.78   46.55    33.76   38.68   31.64   0.948    NS            0.219   NS
   8h-A      22.40             23.38   11.35   30.15    20.26   21.33   21.63   0.052    NS            1.717   NS
   1.5h-A    15.94             8.09    13.54   13.61    14.55   15.25   11.75   3.172    0.116         2.117   NS
  LF                                                                                                           
   24h-A     31.02             20.76   29.00   38.00    29.65   30.34   29.25   1.026    NS            1.241   NS
   12h-A     23.15             14.79   15.99   23.03    23.85   23.50   17.94   2.142    NS            0.018   NS
   8h-A      24.38             11.74   11.28   15.81    18.18   21.28   12.95   2.199    NS            2.031   NS
   1.5h-A    9.27              6.77    12.33   10.12    6.35    7.81    9.74    1.401    NS            0.185   NS
  HF                                                                                                           
   24h-A     62.47             24.21   30.45   42.66    35.59   49.03   32.44   1.170    NS            1.314   NS
   12h-A     38.30             20.22   16.52   21.89    34.08   36.19   19.55   3.440    ***0.083***   1.211   NS
   8h-A      22.78             16.29   13.50   11.87    14.90   18.84   13.89   1.065    NS            1.861   NS
   1.5h-A    13.45             5.53    3.74    8.55     4.95    9.20    5.94    2.947    0.154         3.345   ***0.093***
  LF/HF                                                                                                        
   24h-A     32.88             14.76   11.37   7.33     15.48   24.18   11.15   4.222    **0.033**     9.487   **\<0.001**
   12h-A     15.90             24.65   7.34    8.31     10.68   13.29   13.43   0.079    NS            1.407   NS
   8h-A      11.69             9.97    12.10   6.25     19.31   15.50   9.44    1.242    NS            1.233   NS
   1.5h-A    10.80             10.41   12.23   6.91     9.03    9.91    9.85    0.018    NS            0.535   NS

Amplitudes expressed as a percentage of MESOR, P-values adjusted for multiple testing, considering 6 different frequency regions (ULF01, ULF02, ULF03, VLF, LF, and HF). Based on results from [Tables 1A, 1B and 2](#tbl0005){ref-type="table"}, significant results were anticipated to be found in the ULF rather than in other spectral regions. NN: normal-to-normal intervals; β: slope of fractal scaling; TF: total spectral power; ULF, VLF, LF and HF: spectral power in ultra-low, very low, low, and high frequency regions of the spectrum. Non-sinusoidal waveform may occasionally be associated with overfit (A \> 100%).

###### 

Characteristics of model of 4 anticipated components fitted to some HRV endpoints compared between astronauts whose HR did or did not decrease in space.[\*](#tblfn0020){ref-type="table-fn"}

Table 4

                                    Control (Before flight)   ISS03                                                                                                   
  ------------------- ------------- ------------------------- --------- -------- -------- ------------ ------------ -------- --------- -------- -------- ------------ ----
  MESOR               NN-interval   837.1                     102.1     1021.9   143.6    −2.35        NS           904.1    97.6      1002.0   134.5    −1.31        NS
  TF                  4674.1        1216.8                    12924.0   2750.1   −6.90    **0.0005**   5040.0       1631.2   10287.5   3043.6   −3.66    **0.0320**   
  ULF                 2686.4        798.7                     7229.4    1661.5   −6.09    **0.0015**   2893.6       1138.2   5478.2    2099.0   −2.60    NS           
  ULF01               971.8         443.0                     2161.8    632.8    −3.47    **0.0425**   1744.5       1054.8   2889.3    1064.6   −1.57    NS           
  VLF                 1337.1        337.0                     4536.4    1425.1   −6.02    **0.0015**   1431.4       348.7    3687.2    894.7    −6.06    **0.0015**   
  LF                  572.7         220.4                     1067.8    268.3    −3.08    0.0760       575.4        291.2    853.1     302.4    −1.37    NS           
  HF                  92.4          45.4                      174.6     23.4     −2.91    0.0985       122.7        70.5     137.3     56.9     −0.31    NS           
                                                                                                                                                                      
  24-hour Amplitude   TF            1182.7                    493.5     6430.0   2363.0   −6.05        **0.0015**   3120.4   2248.0    4525.0   3652.8   −0.76        NS
  ULF                 1066.3        570.5                     5055.7    1652.0   −6.01    **0.0015**   2947.5       1944.6   3891.7    3382.3   −0.57    NS           
  ULF01               566.9         444.9                     2885.1    974.0    −5.41    **0.0030**   2322.2       1631.2   2698.9    2172.9   −0.31    NS           
  VLF                 407.2         220.1                     2514.0    1794.6   −3.33    0.0520       580.3        385.9    2149.4    1842.9   −2.32    NS           
  LF                  116.7         84.9                      297.9     209.0    −2.05    NS           126.6        93.9     377.4     329.6    −1.98    NS           
  HF                  30.8          34.3                      88.8      41.3     −2.32    NS           61.5         63.7     65.0      58.0     −0.08    NS           
                                                                                                                                                                      
  12-hour Amplitude   TF            1255.5                    1006.9    6072.5   7806.4   −1.75        NS           2558.4   1965.8    5357.6   1545.4   −2.17        NS
  ULF                 1006.0        784.8                     5433.8    6761.2   −1.86    NS           2601.2       1900.6   4454.7    2538.6   −1.29    NS           
  ULF01               575.4         512.8                     3333.7    2524.9   −2.99    0.0870       2054.6       1686.9   3336.2    2088.9   −1.03    NS           
  VLF                 287.0         219.6                     1662.6    964.5    −3.85    **0.0246**   385.1        184.9    1731.1    1259.6   −3.00    0.0851       
  LF                  73.6          59.9                      214.6     110.8    −2.69    NS           86.3         56.3     231.2     209.1    −1.82    NS           
  HF                  20.2          25.2                      53.7      21.8     −1.99    NS           42.3         33.2     34.0      32.3     0.37     NS           
                                                                                                                                                                      
  8-hour Amplitude    TF            924.6                     503.4     5448.3   4121.8   −3.11        0.0720       2105.5   1417.6    3907.8   3526.6   −1.22        NS
  ULF                 898.5         423.2                     4904.8    3394.5   −3.34    0.0510       1834.5       1428.1   4036.8    2549.7   −1.80    NS           
  ULF01               545.8         258.6                     3025.5    1192.0   −5.64    **0.0025**   1507.4       1222.9   2962.5    1889.2   −1.49    NS           
  VLF                 251.7         164.4                     847.5     445.7    −3.27    0.0570       455.0        247.3    1133.0    633.0    −2.57    NS           
  LF                  111.0         72.6                      229.6     150.4    −1.75    NS           87.4         29.8     150.0     93.6     −1.70    NS           
  HF                  16.3          16.9                      32.1      7.5      −1.53    NS           28.9         23.8     18.4      17.8     0.67     NS           
                                                                                                                                                                      
  90-min Amplitude    TF            154.9                     105.0     1063.1   549.0    −4.55        **0.0095**   532.7    301.3     872.3    738.1    −1.09        NS
  ULF                 117.9         57.5                      790.4     292.0    −6.32    **0.0010**   442.4        202.9    303.2     84.7     1.12     NS           
  ULF01               124.3         82.8                      801.6     155.6    −9.28    **0.0001**   427.6        214.8    303.0     74.2     0.95     NS           
  VLF                 126.0         77.2                      629.5     554.7    −2.56    NS           236.3        121.3    544.1     565.3    −1.48    NS           
  LF                  38.6          31.7                      73.2      33.1     −1.57    NS           48.3         28.6     86.4      63.8     −1.36    NS           
  HF                  6.0           3.7                       19.4      9.1      −3.39    **0.0470**   12.9         14.8     12.8      9.6      0.04     NS           

NN: Normal-to-normal inter-beat interval; TF: Total spectral power; ULF: Ultra low frequency spectral power (0.0001--0.003 Hz).

ULF01: ULF band-1 (0.0001--0.0003 Hz); VLF: very low frequency spectral power (0.005--0.02 Hz).

LF: low frequency spectral power (0.04--0.15 Hz); HF: high frequency spectral power (0.15--0.40 Hz).

P-values adjusted for multiple testing, using Bonferroni inequality (considering 5 tests per endpoint: MESOR, amplitude of each of 4 anticipated components).

MESOR: Midline Estimating Statistic Of Rhythm, a rhythm-adjusted mean.
