Sex Diferences in Post‑exercise Fatigue And Function in Myalgic Encephalomyelitis/chronic Fatigue Syndrome Part 1

To assess biobehavioral sex differences in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) utilizing a low-burden exercise protocol, 22 females and 15 males with ME/CFS and 14 healthy controls underwent two six-minute walk tests. Fifteen daily assessments were scheduled for fatigue and function ratings and heart monitoring. Six-min walk tests were conducted on days 8 and 9. The ME/ CFS group showed high self-report fatigue and impaired physical function, whereas healthy controls did not show fatigue or function abnormalities. In patients, no significant post-exercise changes were found for heart rate variability (HRV); however, heart rate decreased in ME/CFS males from Day 14 to Day 15 (p=0.046). Female patients showed increased fatigue (p=0.006) after the initial walk test, but a downward slope (p=0.008) in fatigue following the second walk test. Male patients showed a decrease in self-report work limitation in the days after exercise (p= 0.046). The healthy control group evidenced a decrease in HRV after the walk tests from Day 9–14 (p=0.038). This pilot study did not confirm hypotheses that females as compared to males would show slower exercise recovery on autonomic or self-report (e.g. fatigue) measures. A more exertion-sensitive test may be required to document prolonged post-exertional abnormalities in ME/CFS.

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Abbreviations 

AIC Akaike information criteria

AR(1) First-order autoregressive 

CS Compound symmetry 

ECG Electrocardiogram 

FSS Fatigue Severity scale 

HR Heart rate 

HRV Heart rate variability 

ME/CFS Myalgic encephalomyelitis/chronic fatigue syndrome 

PEM Post-exertional malaise 

PFS SF-36 physical function subscale 

RMSSD Root mean square of successive differences 

TOEP Toeplitz 

UN Unstructured 

The U.S. United States

Post-exertional malaise (PEM) which refers to prolonged symptom fare-ups after physical activity is a debilitating core symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)1. PEM may lead to increased symptoms of pain and fatigue2–6, abnormal cardiopulmonary responses to exercise7–9, and negative changes in cognitive function10,11. PEM-related behavioral impacts in ME/CFS occur most prominently during the post-exertion recovery period. In a study of self-report PEM following a maximal exercise test12, 85% of healthy controls indicated full recovery within 24 h, in contrast to 0% of ME/CFS patients. Although the entire control group recovered within 2 days, 60% of CFS patients reported that it took 5 or more days to fully recover from the test. Furthermore, in an unpublished finding from a ME/CFS observational study13, a significant sex difference was found in patient-reported PEM, indicating that PEM was of significantly longer duration (ranging from several hours to several days) in females than males (p=0.004). By comparison, overall fatigue intensity, a cardinal symptom of ME/CFS, was not significantly different between females and males.

Post-exercise cardiac autonomic abnormalities in ME/CFS have been found in a two-day repeat maximal exercise test in comparison to healthy controls14,15. The recovery period after the 2-day repeat exercise protocol was more likely to reveal pathophysiological changes2–4,9  apparently because sustained PEM, initially triggered during the first test was then exacerbated during the 2nd test. In healthy controls, full recovery occurs between test 1 and test 214,15. In a recent exercise study16, 16 ME/CFS patients and 10 healthy controls underwent a submaximal warm-up followed by cardiopulmonary exercise testing on two consecutive days. A significant group effect was identified for lower post-exercise heart rate recovery (HRR) in ME/CFS patients. Furthermore, in a case-control study in ME/CFS, a submaximal bicycle exercise test resulted in reduced parasympathetic reactivation during recovery, i.e. slower HRR and lower heart rate variability (HRV) in the ME/CFS group.17

Given the high subject burden and logistical challenges of maximal exercise tests, this pilot feasibility study utilized a modest burden six-minute walk test to potentially trigger post-exercise abnormalities in ME/CFS as evidenced by fatigue elevations and cardiac autonomic abnormalities16,17. Such submaximal exercise testing may be able to distinguish ME/CFS from healthy controls and expand the pool of research participants in this population to include debilitated patients who may not be able to perform a maximal exercise test18. We hypothesized that two high-effort six-minute walk tests (preceded by 30 seconds of fatiguing knee squats) conducted on consecutive days in ME/CFS subjects would show greater adverse impacts and slower recovery to resting baseline in females as compared to males concerning cardiovascular autonomic functioning and symptom resolution. A healthy control group was included to compare the post-exercise trends in autonomic and behavioral measures to ME/CFS cases. Both in-person and home-based participation were utilized in this study.

Methods

Participant recruitment. The baseline target sample size of 40 enrolled participants was intended to achieve, after 20% expected attrition, an endpoint sample of 32 (11 males, 21 females), as 70–80% of ME/CFS patients are female19. The shift to remote visits did not change our recruitment targets.

Preliminary data (N=73) from the PI's laboratory indicated that self-report PEM scores at resting baseline  (frequency x severity ratings of PEM) were positively correlated (p<0.05) with fatigue ratings taken immediately after and 10 min after completion of a standard low exertion six-minute walk test. Fatigue scores at baseline increased at both 10- (p= 0.013) and 20-min (p= 0.005) after completion of the walk test. By comparison, in healthy subjects, the six-minute walk test is associated with lower post-walk fatigue20. This preliminary data suggests that the abnormal symptom exacerbations characteristic of PEM can be provoked and confirmed by patients after a brief, low-effort exercise task as proposed.

Recruitment in the United States (U.S.) began in September 2017 and ended in February 2022. The study initially required in-person visits (n1=24); however, in later years, only remote visits (n2=28) were conducted. In-person subjects were recruited via local advertising, and remotely enrolled participants were reached via national advertising. Targeted participant recruitment methods included internet advertising to large patient organizations (e.g. Health Rising, Solve ME/CFS Initiative) and referrals from the CFS-specialized practice of Dr. Susan Levine which was local to the study site. This was a convenience sample.

Home-based study participation was started in 2019 due to the slow pace of recruitment and later continued with the advent of pandemic restrictions from 2020 to 2022. With home participation, the study was more likely to recruit patients who were disabled and homebound21. For healthy adult subjects, recruitment was done solely via internal notices posted on Stony Brook University's weekly online announcements.

Initial screening. The initial screening of prospective participants for study eligibility was conducted by the project nurses (PB, MM) utilizing a validated phone interview. Selection criteria were: (a) age between 21 and 6523,24; (b) Fukuda-based CFS symptoms including 6 months of medically unexplained, debilitating fatigue plus 4/8 secondary symptoms, i.e., memory or concentration differences, unrefreshing sleep, sore throats, headache, muscle pain, joint pain, tender lymph nodes, post-exertional malaise; and (c) absence of exclusionary illnesses. To meet CFS symptom criteria, each of the 4 qualifying symptoms had to be endorsed with a frequency of “sometimes” or greater and an intensity of “moderate” or higher. The phone interviewer identified medical exclusions of fatigue attributable to self-report medical conditions, e.g., untreated hypothyroidism24. In addition, individuals were excluded if they were taking heart-altering medication (e.g. beta-blockers and antidepressants). Exclusionary psychiatric disorders included any psychosis, or alcohol/substance abuse within two years before illness onset and any time afterward, and current or past depression with melancholic or psychotic features within 5 years before onset of CFS or anytime afterward 24.

The screening of prospective healthy participants was also conducted by the project nurses (PB, MM)22.  These self-identified healthy individuals were required to be aged 21–65 and were excluded if they reported the presence of a chronic illness, either medical or psychiatric and/or were taking prescription medication for an ongoing illness. All study subjects were considered physically capable of doing the exercise tasks and were willing to wear a heart monitor.

Procedure. The study procedures (Fig. 1) included: (a) participant eligibility phone screening followed by land mailing, completion, and return of the informed consent form and standard questionnaires (pre-study baseline only); (b) mailing and return of heart monitor after data collection was complete; (c) 15 days of online fatigue and functional limitation ratings and home-based HRV monitoring (10 min/day); (d) Staf-guided instructions (in person or via phone call) using a written stepwise protocol to guide each participant through study procedures.

Instructions to participants included attachment of the heart monitor13, use of the online web diary, and conduct of the knee squats and the two walk tests. Participants were asked to abstain from caffeine and vigorous exercise for 24 hours and from eating for 3 hours before the walk tests. These instructions with a demonstration video on how to do self-paced knee squats were given:

Stand with your feet slightly wider than your hips. Toes pointed slightly outward. Your weight should be on your heels and the balls of your feet as if you were pasted to the ground (as if about to sit down). Now look straight ahead and pick a spot on the wall in front of you. Look at this spot the entire time you squat, not looking down at the floor or up at the ceiling. As you squat, keep your upper body straight. Don’t bend forward. Ready. Go ahead.

The 30 s of knee squats, intended to increase exertional impacts, was followed by a maximum effort (“walk as fast as you can”) six-min walk test scheduled for days 8 and 9. The home-based tape-measured walking course consisted of repeated laps on relatively straight paths, i.e. zig-zags allowed. The straightaway section varied in length depending on the house configuration. These repeat physical exercise tasks were intended to trigger PEM, given that post-exertion impacts can result from relatively minor physical effort in ME/CFS individuals18. This study was approved by the Stony Brook University Committee on Research Involving Human Subjects and all participants provided informed consent. All methods were performed according to the relevant guidelines and regulations.

Standard questionnaires. Fatigue severity scale (FSS). This measure of the effect of fatigue on functioning is comprised of nine items rated on a seven-point Likert-type rating scale, where one indicates no impairment and seven indicates severe impairment (score range: 1.00–7.00). In the initial validation study26, internal consistency for the FSS was excellent (Cronbach's α=0.80) and the scale clearly distinguished between patients and controls. The scale, recommended for use in CFS27, showed high alpha consistency in our sample (Cronbach's α=0.87).

SF‑36 physical function subscale (PFS). The PFS28 of the SF-36 measures physical limitations of ill health on a scale of 0 to 100, where 0 indicates limited in all activities, including basic self-care and 100 indicates no limitations. The PFS is composed of ten items and each item is scored based on the limitations perceived by surveyed individuals. Item scores (1, 2, or 3) are summed to obtain a total score. Normative values are available for the PFS for the U.S. population. This subscale has shown good internal consistency (Cronbach’s α≥0.81) in psychometric studies28 and our sample (α=0.88).

Online web diary (Day 1 to Day 15). The online web diary (StudyTrax, Inc., Macon, Georgia) was scheduled daily and directed participants to rate fatigue intensity and 3 types of activity limitations for job and home activities at the end of each study day (work, housekeeping, exercise). Both fatigue intensity and activity limitations were ordinal variables with integer values from 0 (None/No limitation) to 10 (Highest/Major limitation).

Objective measures. Knee squats. Knee squats are a type of resistance training that has been shown to increase perceptions of both effort and fatigue e.g., in healthy populations. Thirty seconds of knee squats were scheduled immediately before each six-minute walk test to increase post-exertion fatigue and potential autonomic dysfunction triggered by the walk tests.

Six‑minute walk test. This is a sub-maximal exercise test of functional capacity which measures the distance walked during a six-minute interval. The test is a useful and reproducible measure of exercise tolerance which does not require expensive apparatus31. To enhance post-exertional impacts, subjects were instructed to “walk as fast as you can.

Heart rate monitor (Day 1 to Day 15). Heart rate variability (HRV) and heart rate (HR) are non-invasive measures of cardiac autonomic function. Both variables were calculated via electrocardiogram (ECG) collected with a research-grade multifunction ambulatory heart monitor (eMotion Faros 180; MegaElectronics, Kuopio, Finland). ECG data were collected at a 500 Hz sampling rate using a 3-lead configuration. Data were exported for analysis in the Kubios HRV analysis suite (version 3) for R-peak detection the visual inspection of artifacts (e.g. non-sinus beats, movement), and HRV calculation. R-peaks were detected using a modified Pan-Tompkins algorithm32,33. Detected artifacts were corrected by replacing artifacts with interpolated values via cubic spline interpolation.

The root mean square of successive differences (RMSSD) was then calculated to estimate HRV. RMSSD reflects beat-to-beat variance in heart rate and is the primary time-domain measure used to estimate the vagally mediated changes reflected in HRV34. To approximate a standard setting, HR data were collected daily by participants at home while sitting in a comfortable chair for ten minutes in the evening between 7 p.m. and 9 p.m. Subjects were instructed to be in “quiet time” (no other activity, including TV) during resting HRV assessment.

This small beeper-size heart monitor was sent to each participant. Proper electrode placement was guided by written and pictorial instructions and confirmed with a staff phone call to each participant. Consistent with recommendations 35, inter-beat intervals recorded for five min (or more) are sufficient to approximate parasympathetic outflow to the heart 36. The first two minutes of data were discarded to provide an acclimatization period.

Data analysis. Daily data for all measures were collected from Day 1 to Day 15 for the HR autonomic measure, fatigue ratings (0–10), and ratings (0–10) of functional limitations regarding work, housekeeping, and exercise limitations. Day 1 to Day 8 were baseline days and Day 9 to Day 15 were walk test and post-walk test days. The “baseline” value was the average for each variable from Day 1 to Day 8. Analyses focused on aggregate baseline data (days 1–8) and the data for each day from Day 9 to Day 15. All outcome variables were treated as continuous variables to analyze linear trends over time.

Piecewise linear mixed effect models37 were used to analyze the daily longitudinal data (HRV, HR, fatigue intensity, and three types of activity limitations). To compare the differences in daily measurements between females and males in the ME/CFS group, fixed effects adjusted in the models were for gender, a time variable, a time spline variable, an interaction term between gender and time, and an interaction term between gender and time spine variable. Time was a continuous variable with integer values 0 to 7 to represent the baseline (0) and time points from Day 9 (1) to Day 15 (7). Time spline was also a continuous variable with values 0 to 7 to divide time into 2 segments. The breakpoint in the linear trend was decided by using the smallest model goodness-of-ft statistics 38. Similarly, piecewise linear mixed effect models with adjustment for the treatment group were utilized to compare daily data between ME/CFS and health control groups. Based on Akaike Information Criteria (AIC), the covariance structure to model correlation among longitudinal measurements from the same patient is selected from Compound Symmetry (CS), first-order autoregressive (AR(1)), Toeplitz (TOEP), and Unstructured  (UN). Statistical analysis was performed using SAS 9.4 (SAS Institute Inc., Cary, NC) and the significance level was set at 0.05.

Results

Participant characteristics. Of the 118 phone-screened individuals, 67 (56.8%) were excluded for not meeting entry criteria as follows: subthreshold CFS symptoms (33/67), self-report exclusionary medical/psychiatric illness (22/67), and other factors, e.g. age or BMI out of range (12/67). Fify-one individuals were enrolled and 9 (17.3%) were lost to follow-up. Most participants (Table 1) were in their 40s, white (90.3%), and female.  The majority of ME/CFS participants were ill for over a decade. Fourteen subjects were healthy controls. For the ME/CFS group, standard questionnaires showed high fatigue severity and impaired physical function. Healthy controls did not show fatigue or function abnormalities.

Symptom presentations of ME/CFS participants were consistent with the findings of a large sample factor analytic study of diagnostic criteria in ME/CFS39. This study identified these apparent core symptom dimensions: cognitive dysfunction and post-exertional malaise (each endorsed by 90+%) and sleep dysfunction (79%). Our screening data also showed high levels of endorsement for impaired memory or concentration (93%; N=40), post-exertional malaise (100%; N=43), and unrefreshing sleep (97.7%; N=42). Compliance for 15 days of web diary data collection was 92.1% for female patients, 84.9% for male patients, and 86.7% for healthy controls. Six-min walk test distances for ME/CFS participants averaged over 2 walk tests for males was 342.62 m (SD: 129.12) and for females, 382.28 m (SD: 120.77). Walk test distances were not recorded for healthy controls. For knee squats in the ME/CFS group, the number of squats averaged over two 30-s sessions was 11.25 (SD: 3.58)   for males and 10.86 (SD: 2.79) for females.

Sex differences within the ME/CFS group. Female and male data in the ME/CFS group for pre-walk test baseline and post-walk test days 9–15 are shown in Table 2. Figures 2, 3, and 4 show the estimated linear trends by sex for HRV, HR, and fatigue, respectively, based on piecewise linear mixed-effect models.

No significant changes over time were found for HRV (Table 3); however, heart rate significantly decreased in ME/CFS males from Day 14 to Day 15 (Fig. 3) and the slope change in heart rate at Day 14 was also significant. Female patients showed a significant increase in average fatigue rating from baseline to the first day (Day 9) after baseline which included the initial walk test (Fig. 4). Also, for ME/CFS females, a significant negative slope for fatigue was found from Day 9 to Day 15. In addition, the change in slope for fatigue on Day 9 was significant. No other significant linear trends were found for females. Male patients did not show significant linear trends for fatigue but did show a significant decrease (E=− 0.184; CI − 0.37– − 0.00; p=0.046) in self-report work limitation from the 2nd day after baseline, i.e. Day 10 to Day 15.

Trends in the ME/CFS treatment group and healthy control group. Average parameter values for heart variables and self-report fatigue and functional limitations for ME/CFS and healthy control groups are listed in Table 4. Regression analysis results based on piecewise linear mixed effect models are presented in Table 5.  Figures 5, 6, and 7 show the plots of the estimated linear trends of HRV, heart rate, and fatigue, respectively, from baseline to post-walk test days 9 to 15 for the ME/CFS and healthy control groups.

Based on the analysis of the ME/CFS and healthy control groups (Table 5), HRV (Fig. 5) showed no significant changes in the ME/CFS group; however, the healthy control group showed a significant decrease in HRV after the walk tests from Day 9 to Day 14. No significant changes were found in heart rate. Regarding fatigue (Fig. 7), the ME/CFS group first showed a significant increasing pattern from baseline to Day 9 and then a significant decreasing pattern from post-walk test Day 9 to Day 15. The fatigue slope change in the ME/CFS group (Fig. 7), which occurred on the 1st day after the initial walk test (Day 9) was also significant.

The ME/CFS and healthy control groups showed significantly different trend slopes for fatigue from baseline to Day 9. Also, these two groups showed different patterns of slope change on Day 9. Regarding self-report exercise limitation, ME/CFS patients showed a significant change in trend slope at post-baseline (Day 13) (E=− 0.281; CI − 0.53–− 0.03; p=0.029). The ME/CFS and healthy control groups showed different trend slopes for exercise limitation after the Day 13 breakpoint (E=− 0.327; CI − 0.61–− 0.05; p=0.023). In addition, these two groups showed differential changes in slope for exercise limitation ratings at the breakpoint on Day 13 (E= − 0.524; CI − 0.94–− 0.11; p=0.015).

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