The prevalence of five to seven ideal CVH metrics increased from 11.6% in 2007 to 37.9% in 2019. Trend analyses showed that ideal physical activity, ideal total cholesterol, ideal blood pressure, and ideal fasting glucose increased from 2007 to 2019, while ideal fruit and vegetable intake, ideal smoking, and ideal body mass index decreased from 2007 to 2019. Five to seven ideal CVH metrics, 0–7 ideal CVH metrics, and 0–14 poor, intermediate, and ideal CVH metrics increased from 2007 to 2019. In the adjusted logistic regression analysis, older age decreased the odds of having 5–7 ideal CVH metrics in all three study years. Male sex increased the odds, and employment decreased the odds of 5–7 ideal CVH metrics in 2007, and urban residence increased the odds of 5–7 ideal CVH metrics in 2014 and decreased the odds in 2019. The proportion of meeting 5–7 ideal CVH metrics increased in Bhutan. Primary and secondary prevention programmes must be strengthened to improve CVH in Bhutan, considering identified associated factors.The study aimed to estimate the prevalence, distribution, and correlates of ideal cardiovascular health (CVH) among individuals (20–69 years) across three cross-sectional surveys in 2007, 2014 and 2019 in Bhutan. Cross-sectional data were analysed from 9712 individuals  who participated in the 2007, 2014 or 2019 Bhutan STEPS surveys, had complete measurement of CVH metrics, and had no history of a cardiovascular disease in 2014 and 2019. Ideal CVH measures included non-smoking, healthy diet, physical activity, body mass index (< 23 kg/m CVDs contribute to 28% of all death in 2016 in Bhutan, a lower middle-income country in south central Asia2. “Non-communicable diseases (56%) and injuries (a staggering 19%) account for over three-quarters of all deaths in Bhutan and the probability of dying prematurely (before the age of 70) from one of the four main NCDs is one in five”3. CVD risk factors in Bhutan include a “now high prevalence of overweight and obesity being observed among adult populations , two thirds of the population do not consume sufficient fruit and vegetables and smoke, and smokeless tobacco is widely used (34% in men and 14% in women)”3. These CVD risk factors often cluster together, increasing the risk of developing CVDs, and should be prioritized in the prevention of CVDs5.Globally, an “estimated 17.9 million people died from cardiovascular diseases (CVDs) in 2019, representing 32% of all global deaths”, of which more than three-quarters occurred in low- and middle-income countries7. Using these seven metrics, the CVH status of the population of the population can be defined as ideal , intermediate  or poor 8. Having a higher number of ideal CVH metrics has been shown to be protective against “the risk of all-cause and CVD-related mortality, incident cardiovascular events, lower prevalence and incidence of non-CVD outcomes such as cancer, depression, and cognitive impairment”9. In more recent studies, for example, among a middle-aged Finnish population10 and a Chinese adult population, the CVH score decreased the risk of CVD mortality11. We could not find any reports on ideal CVH in Bhutan.In an effort to prevent the development of CVDs, the American Heart Association (AHA) conceptualized “ideal cardiovascular health (CVH)”, including seven ideal health factors and behaviours, including blood pressure, blood glucose level, total cholesterol, level of smoking, physical activity, body mass index, and nutritional intake8. Few reports have been found on CVH in East and South Asian low- and middle-income countries. Several studies in China found, e.g., in Shandong (18–69 years) 0.05% had all 7-ideal metrics12, in rural Northwest China (20–80 years) 0.0%13 and in rural China (≥ 35 years), 0.1% had all 7 ideal CVH metrics14. In a nationally representative sample in China (≥ 20 years), 33.0% had 5–7 ideal CVH15. In South Asia, in Nepal (15–69 years), 51.6% had 5–7 ideal CVH metrics16, in semi-urban Western Nepal (≥ 25 years), 14.3% had 6 or 7 ideal metrics5, and in urban India (20–75 years), ideal < 0.1% had 7 ideal metrics and 7.1% had ≥ 6 ideal metrics17. Globally, dietary pattern (12.1%) had the lowest prevalence of CVH status, followed by blood pressure (BP) (34.6%), body mass index (BMI) (40.3%), physical activity (40.6%), total cholesterol (TC) (51.7%), fasting blood glucose (FPG) (67.7%), and smoking (69.1%)8.Globally, mainly in high-income countries, 19.6% of participants had ideal  CVH18. In a trend study over ten years in Iran, the prevalence of 5–7 ideal CVH metrics slightly reduced from 22.7% in 2007 to 19.5% in 2016, the mean CVH metrics increased from 4.7 in 2007 to 5.0 in 201619. In a trend study among adults in USA over seven years the prevalence of the ideal CVH score significantly increased from 3.89 in 2011 to 3.96 in 201720, and a trend study over 20 years in France found a decrease in ideal CVH21.We could not find any trend study on ideal CVH in East and South Asian countries, with the exception of a study in North China, which however, does not report the total ideal CVH metrics22, younger age23, ethnicity22, higher education23, and rural residence25. The study aimed to estimate the prevalence and associated factors of ideal CVH among individuals (≥ 20 years) in three cross-sectional surveys in 2007, 2014 and 2019 in Bhutan.Sociodemographic factors associated with ideal CVH may include female sex26 with complete CVH metrics measurements and no history of CVD in 2014 and 2019 were analyzed; the overall response rate was > 93% in 2014 > 96% in 201929. “A multi-stage stratified sampling process was carried out to randomly select participants from the target population. An individual within the age range of the survey” was selected per household 29. In 2007 the study was restricted to the capital city of Thimphu, and in 2014 and 2019 the whole country of Bhutan. We restricted our data analyses to persons 20–69 years and those with no missing data on “smoking status, BMI, PA, diet, total TC, FBG and BP” measurements. The Research Ethics Board for Health (REBH), Bhutan, approved the study and written informed consent was obtained from all participants29. All methods were performed in accordance with the relevant guidelines and regulations.Secondary data from three cross-sectional STEPwise approach to surveillance (STEPS) surveys in Bhutan in 2007, 2014, and 201926. Anthropometric measurements were taken with a portable digital weighing scale (SECA 843), constant tension Myotape tape (SecaTMbrand)29. Prior to taking blood pressure measurements, participants were asked to sit quietly and rest for 15 min with legs uncrossed. Three readings of systolic and diastolic blood pressure were obtained, with participants resting for three minutes between each reading. Of the three blood pressure measurements using “Omron BP apparatus automatic blood pressure monitor”29; the last two readings following recommendations by WHO were averaged26. “Blood glucose, and total cholesterol were measured in peripheral (capillary) blood at the data collection site using dry chemical methods, biochemical analysis with a Hitachi 912 bio-analyzer”29.Data collection followed the “WHO three STEPS methodology: step 1 included administration of a structured questionnaire  step 2 consisted of blood pressure and anthropometric measurements, and step 3 included biochemical tests (blood glucose and blood lipids)”7.Poor, intermediate and ideal CVH levels for “smoking, BMI, PA, diet, TC, BP, and FBG” were evaluated following modified AHA definitions, exact AHA classifications are given in bracketsSmoking status: ≥ 20 years, smoking is defined as poor if current smoker (in the past 12 months), and ideal if self-report not past 12-month (current) smoker .Body Mass Index (BMI) (kg/m2): “ ≥ 20 years, BMI is defined poor if ≥ 25 kg/m2, intermediate as 23.0–24.9 kg/m2, and ideal BMI is < 23.0 kg/m2”30 .Healthy diet: adults ≥ 20 years, poor healthy diet is defined as “ < 2 servings of fruit and vegetables (FV)/day, intermediate as 2–< 4.5 FV/day, and an ideal diet as ≥ 4.5 FV servings/day”31; .Physical activity (PA): adults ≥ 20 years, “Poor = None, Intermediate =  < 600 MET mins/week, 600–< 1500 MET mins/week, and ≥ 1500 MET mins/week, based on the Global Physical Activity questionnaire32. .Poor total cholesterol (TC) is classified as adults ≥ 20 years, “poor is TC ≥ 6.3 mmol/L (≥ 240 mg/dL), intermediate is TC 5.2–6.2 mmol/L (200–239 mg/dL), and ideal TC is < 200 mg/dL” .Fasting blood glucose (FBG): adults ≥ 20 years, poor FBG is defined as “glucose ≥ 7.0 mmol/L (≥ 126 mg/dL), intermediate is glucose 5.6–6.9 mmol/L (100–125 mg/dL) or treated to < 100 mg/dL, and ideal is < 5.6 mmol/L < 100 mg/dL and without any glucose-lowering medication” [Adults ≥ 20 years].Blood pressure (BP): adults ≥ 20 years, poor is defined as “BP ≥ 140/ ≥ 90 mmHg, intermediate is systolic BP 120–139 mmHg or diastolic BP 80–89 mmHg or treated to BP < 120/ < 80 mmHg, and ideal BP is defined as BP < 120/ < 80 mmHg and without any antihypertensive medication” [Adults ≥ 20 years].The seven CVH metrics were coded as 1 = ideal and 0 = not ideal, summed, and classified into 0–2, 3–4, and 5–7 ideal CVH metrics.26.In STEPS surveys 2014 and 2019 but not in 2007, “history of CVDs included self-reported coronary heart disease; angina, also called angina pectoris; a heart attack ; stroke; any kind of heart condition or heart disease (other than the ones I just asked about) ”24.Sociodemographic covariates included age (years), sex , education in years, number of adult household members, employment, and residence statusP values < 0.05 were considered significant and missing values were excluded from the analysis.All statistical analyses were conducted with “STATA software version 14.0 ”. “Analysis weights were calculated by taking the inverse of the probability of selection of each participant. These weights were adjusted for differences in the age-sex composition of the sample population as compared to the target population”. Taylor linearization methods were utilized to account for complex study design. Descriptive statistics are used to describe CVH metrics . Chi-square tests were used to test for differences in proportion. The significance of linear trends was analysed by using study year as categorical variable in logistic, linear, and ordinal regression analyses depending on the outcome variable. Logistic and linear regressions were used to assess the associations between sociodemographic factors and meeting 5–7 CVH metrics and continuous CVH metrics, overall and stratified by sex. Covariates in the multivariable logistic and linear regression models were age group, sex, educational level, number of adult household members, work and residence status. Cross-sectional data were analysed from 9712 individuals , 2187 in 2007, 2588 in 2014, and 4,937 in 2019. Further sociodemographic characteristics of the sample by study year are described in Table Trend analyses showed that ideal physical activity, ideal total cholesterol, ideal blood pressure, and ideal fasting glucose increased from 2007 to 2019, while ideal fruit and vegetable intake, ideal smoking, and ideal body mass index decreased from 2007 to 2019. Five to seven ideal CVH metrics, 0–7 ideal CVH metrics and 0–14 poor, intermediate, and ideal CVH metrics increased from 2007 to 2019. The prevalence of five to seven ideal CVH metrics increased from 11.6% in 2007 to 37.9% in 2019, the seven-mean ideal CVH metrics increased from 2.9 in 2007 to 4.2 in 2019, and the 12-mean ideal CVH metrics increased from 8.7 in 2007 to 10.2 in 2019  in Bhutan increased from 11.6% in 2007 to 37.9% in 2019, and the mean CVH metrics increased from 2.9 in 2007 to 4.2 in 2019. A similar increase in the ideal CVH score was found in a trend study in the USA 15, but lower than in Nepal 16. The proportion of ideal CVH metrics  , was similar to in previous studies, such as in urban India 17, Shandon in China 12, in rural area Northwest China all 7 ideal metrics (0.0%)13.The prevalence of ideal CVH  (ranging from 11.6% in 2007 to 37.9% in 2019), were similar to global estimates, mainly in high-income countries, ideal CVH  (19.6%) CVH8, this survey found that TC (92.7% in 2019), smoking (89.6% in 2019), and FGP (91.9% in 2019) had the highest proportion of ideal CVH status. A healthy diet (16.2% in 2019) had the poorest prevalence of ideal CVH status in this study which compares with the poorest global estimates8. The proportion of ideal fruit and vegetable consumption compares with global rates in low- and middle-income countries (18.0%)33. The estimates of ideal PA (84.7% in 2019) in this study were higher than global estimates of PA (40.6%) and ideal BMI (34.3% in 2019) are lower than global figures of ideal BMI (40.3%)8. Ideal blood pressure significantly decreased from 35.8% in 2007 to 30.1% in 2019 and poor blood pressure or hypertension significantly increased from 19.9% in 2007 to 26.6% in 2019, which is higher than global estimates in low- and middle-income countries (17.5%)34. Previous research in Bhutan also found a high prevalence of poor diet, overweight, and high blood pressure among adults calling for expanded supplementation and dietary interventions3.Like the three best global estimates23, ideal CVH was higher among younger age groups (20–34 years) and among men in 2007. Some previous research23 showed an association between higher education and ideal CVH metrics, while we only found this result, in particular among women, in 2019. Other research25, found an association between rural residence and ideal CVH metrics, which was also found in this study in 2019, while we found higher ideal CVH metrics in residents of urban than rural areas in 2014.Consistent with previous research35. Diabetes and raised total cholesterol may have reduced due to the universal health coverage, including the early diagnosis and management of NCDs in Bhutan3. To improve CVH in Bhutan, multifactorial interventions are needed36, targeting the promotion of body weight control, smoking cessation, healthy diets, and screening and control of high levels of blood pressure5.Results may inform Bhutan’s CVH promotion policy. The increase in ideal physical activity may be attributed to the “implementation of community wide public education and awareness campaigns for physical activity”, including a nationwide “Move for Health Campaign” is conducted routinely and open-air gyms in Thimphu and a number of other districts9. Another limitation was that the history of any previous CVD was not evaluated in the 2007 and only in the 2014 and 2019 survey.In addition to the use of large representative survey samples and standardized assessment measures, the study limitations included the cross-sectional design and self-report of some of the data. In addition, our healthy diet measure only consisted of one item (fruit and vegetable consumption) and not as in the original AHA healthy diet definition “”The proportion of meeting 5–7 ideal CVH metrics increased in Bhutan. Primary and secondary prevention programmes should be strengthened to improve CVH in Bhutan, considering identified associated factors. Future research may include more comprehensive measures on a healthy diet.


The accumulation of iron may contribute to Alzheimer’s disease (AD) and other tauopathies. The iron chelator desferrioxamine slows disease progression in AD patients. However, desferrioxamine requires injection, which is inconvenient and may hinder compliance. We therefore tested an oral iron chelator, desferasirox (Exjade), in transgenic animal models. Tg2576 mice overexpress the mutant human APP protein and produce the Aβ peptide. JNPL3 mice (Tau/Tau) overexpress the mutant human tau protein. Crossing these produced APP/Tau mice, overexpressing both APP and tau. Treating the three models with 1.6 mg deferasirox thrice weekly from age 8 to 14 months did not affect memory as measured by contextual fear conditioning or motor function as measured by rotarod, but tended to decrease hyperphosphorylated tau as measured by AT8 immunohistochemistry and immunoblotting. Deferasirox might act by decreasing iron, which aggregates tau, or directly binding tau to inhibit aggregation. Bo. Bo3+ biognition ,20. ThusBy decreasing the level of iron in animal models, iron-induced tau phosphorylation was reduced . Iron chTransgenic mouse models were bought (Taconic Farms) and bred at The Chinese University of Hong Kong  in standard housing conditions, and the University’s Animal Experimentation Ethics Committee approved the study. APP transgenic mice (Tg2576), which are hemizygous for the human β-amyloid precursor protein gene with the Swedish mutation K670N/M671L) (APPswe), overexpress Aβ. They were bred on the F1 progeny of SJL/JcrNTac and C57BL6/NTac strains. Tau/Tau transgenic mice (JNPL3) were used as a tauopathy model. They are homozygous for the 4R0N isoform of the human microtubule-associated protein tau with the P301L mutation. Crossing JNPL3 with Tg2576 produced Tau/APP mice with one copy of the tau transgene and one copy of the APP transgene, as described previously ,24. We eL  and then increased at 1 rpm/9 s, to a maximum of 40 rpm. The time between the beginning of the acceleration and the falling of each mouse was recorded, thrice before treatment and thrice after treatment. The difference in the averages before vs. after treatment was calculated for each mouse.Contextual fear conditioning (CFC) involves placing an animal in a new environment, giving an aversive stimulus, observing the response, waiting some time, and then putting the animal in the same environment but without an aversive stimulus to observe whether the animal exhibits fear (freezing its motion), indicating memory of the association of that environment with the aversive stimulus. Each mouse was placed in a chamber, and a tone was played, followed by an electric shock. The next day, context memory was tested by returning each mouse to the chamber and recording the time spent frozen. Cue memory was tested by moving the mouse to a novel chamber and recording the time spent frozen after the playing of the same tone as the previous day.The distribution and phosphorylation of tau in the brains of transgenic mice  was analyzed by immunohistochemistry. Briefly, coronal paraffin sections were dewaxed, rehydrated, boiled in citrate buffer , rinsed in 3% hydrogen peroxide (endogenous peroxidase quenching), rinsed with phosphate-buffered saline (PBS), blocked with 10% normal goat serum, and incubated overnight with mouse anti-tau-phospho-Ser202/Thr205  at room temperature. Sections were rinsed with PBS, incubated with DAKO Envision Labelled Polymer (Cat# K4001), rinsed, developed by DAB , rinsed with water, counterstained with Harris Hematoxylin, mounted, and examined by light microscope with a digital camera (Zeiss).Two AT8 stained sections, about 80 μm apart, were quantitated. For each section, four fields were randomly selected from the medulla (about −5.68 to −6.00 mm from bregma) at 40× magnification based on the highest intensity of AT8 signals. Pixels within the color threshold were counted using SigmaScan Pro 5.0.4, 6 mM dithiothreitol, 0.02% sodium azide, Protease Inhibitor Cocktail Set III  at 1:100 dilution, and phosphatase inhibitor cocktail), centrifuged 15 min at 13,000× g and 4 °C, and supernatants removed and assayed to normalize sample concentrations. Normalized amounts were centrifuged for 60 min at 100,000× g and 4 °C. Pellets were homogenized in PHF extraction buffer  and centrifuged for 30 min at 100,000× g and 4 °C. Supernatants were transferred to new tubes and incubated with sarkosyl (1%) for 1 h at 37 °C. After centrifugation at 100,000× g for 60 min at 4 °C, pellets were resuspended in Tricine sample buffer . Samples were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and AT8 Western blotting using enhanced chemiluminescence.From each mouse brain, tissue was extracted as previously described . Brainstp < 0.05. Data were presented as mean ± standard deviation, and differences among groups were analyzed by independent sample t tests. The effect of the drug on mortality was analyzed by Fisher’s Exact Test.Results were analyzed by SPSS Statistics (v20). Differences were considered significant if A total of 106 mice entered treatment . Only a Contextual fear conditioning (CFC) tests measure memory of a location (context) or a sound (cue), expressed as the percentage of time spent frozen. By age 14 months, female Tau/Tau and Tau/APP mice had deteriorated to such a degree that they were unfit for CFC, thus only males were studied . For APPp = 0.33, and CFC cue memory changed −16 ± 17% without deferasirox and −6.2 ± 37% with deferasirox, p = 0.47. Between the start and end of treatment in Tau/APP mice, CFC context memory changed −18 ± 29% without deferasirox and −8.9 ± 35% with deferasirox, p = 0.56, and CFC cue memory changed −33 ± 28% without deferasirox and −8.9 ± 20% with deferasirox, p = 0.062. Between the start and end of treatment in APP mice, CFC context memory changed −11 ± 20% without deferasirox and −19 ± 22% with deferasirox, p = 0.43, and CFC cue memory changed +2.8 ± 24% without deferasirox and −12 ± 25% with deferasirox, p = 0.19.Between the start and end of treatment in Tau/Tau mice, CFC context memory changed −9.4 ± 27% without deferasirox and 2.9 ± 25% with deferasirox, Rotarod tests measure motor ability and coordination. At age 14 months, female Tau/APP mice had deteriorated to such a degree that they were not fit for rotarod tests, thus only males were studied . For Taup = 0.94. Between the start and end of treatment in Tau/APP mice, the rotarod times changed +11 ± 43 s without deferasirox and +11 ± 45 s with deferasirox, p = 0.96. Between the start and end of treatment in APP mice, the rotarod times changed +3.5 ± 29 s without deferasirox and +3.5 ± 37 s with deferasirox, p = 1.00.Between the start and end of treatment in Tau/Tau mice, the rotarod times changed −16 ± 65 s without deferasirox and −13 ± 30 s with deferasirox, p = 0.22. In Tau/APP mice, NFTs occupied 4.5 ± 2.6% without deferasirox treatment and 3.8 ± 3.4% with deferasirox: a 15% decrease, p = 0.57.The area occupied by NFTs in mouse brains  was analyzed by immunohistochemistry using monoclonal antibody AT8, which targets Ser202 and Thr205 of the hyperphosphorylated tau in NFTs . In Tau/Only a small number of APP mice were available for study. Thus, analysis of their brains was not undertaken.From each brain, a fraction enriched in paired helical filaments (PHF) was extracted and subjected to AT8 Western blotting to measure accumulation of the 64 kD band of phosphorylated tau . Only map = 0.64. In Tau/APP mice, the level of phosphorylated tau was 26 ± 12 without deferasirox and 12 ± 14 with deferasirox: a 53% decrease, p = 0.03.In Tau/Tau mice, the level of phosphorylated tau was 24 ± 17 (arbitrary units) without deferasirox and 18 ± 17 with deferasirox: a 24% decrease, In the particular conditions of this study, deferasirox treatment of AD and tauopathy models did not demonstrate protection of memory or motor ability. However, there is a possibility that deferasirox decreased hyperphosphorylated tau accumulation, as indicated by decreased hyperphosphorylated tau on Western blots of Tau/APP mice. This is consistent with trends toward decreased hyperphosphorylated tau on Western blots of Tau/Tau mice and in immunohistochemistry of both Tau/Tau and Tau/APP mice.Consistent with a possible protective effect of deferasirox in tauopathy are the trends toward relatively better memory, in both CFC cue and context tests, in both Tau/Tau and Tau/APP mice. However, because the p-values were high and because many comparisons were tested in this study, replication of these findings would be needed.Although not statistically significant, it may be worth noting the trend toward worsening of memory with deferasirox in APP mice. Perhaps deferasirox acts directly or indirectly on tau, but not on Aβ, and removal of iron to levels below the optimum may impair function in the absence of a specific benefit. However, human studies of deferasirox have not led to cognitive decline .If deferasirox does reduce hyperphosphorylated tau levels, there are several possible mechanisms. Iron induces aggregation of hyperphosphorylated tau, thus such an effect might be due to iron chelation . AnotherThese results suggest fruitful directions for future research. Studies of deferasirox in larger numbers of animals should be conducted to determine whether this drug has the potential to treat tauopathies. Epidemiological studies may investigate whether long-term users of deferasirox and other iron chelators display a lower risk of developing tauopathies . Clinical trials in people genetically prone to later develop tauopathy could explore whether iron chelators slow or prevent tau deposition and neurodegeneration.


Results proved that β-cyclodextrin, with relative low accumulation in the body, had good safety. The results will assist further study of the design and safety evaluation of adjuvant β-cyclodextrin and promote its clinical development.β-cyclodextrin has a unique annular hollow ultrastructure that allows encapsulation of various poorly water-soluble drugs in the resulting cavity, thereby increasing drug stability. As a bioactive molecule, the metabolism of β-cyclodextrin is mainly completed by the flora in the colon, which can interact with API. In this study, understanding the in vivo fate of β-cyclodextrin, a LC-MS/MS method was developed to facilitate simultaneous quantitative analysis of pharmaceutical excipient β-cyclodextrin and API dextromethorphan hydrobromide. The established method had been effectively used to study the pharmacokinetics, tissue distribution, excretion, and metabolism of β-cyclodextrin after oral administration in rats. Results showed that β-cyclodextrin was almost wholly removed from rat plasma within 36 h, and high concentrations of β-cyclodextrin distributed hastily to organs with increased blood flow velocities such as the spleen, liver, and kidney after administration. The excretion of intact β-cyclodextrin to urine and feces was lower than the administration dose. It can be speculated that β-cyclodextrin metabolized to maltodextrin, which was further metabolized, absorbed, and eventually discharged in the form of CO Pharmaceutical excipients are defined as any substance in drugs possessing pharmacological inertia, except for active pharmaceutical ingredients (API). The excipient added in the production of API formulations has various functions such as improving tablet performance, ensuring API dose uniformity, changing API release, and masking odor . Excipieβ-CD is a cyclic oligosaccharide containing seven pyranose glucose units, which can be produced by amylose under the action of cyclodextrin glucosyltransferase . β-CD haThe mass spectrometry detection method of HP-β-CD had been reported in our previous study ; howeverβ-cyclodextrin (β-CD) was acquired from Xiya Chemical Industry Co., Ltd. . Dextromethorphan hydrobromide (DM), Ginsenoside Re , and Psoralen  were bought from Chengdu Chroma-Biotechnology Co., Ltd. . The structures of the aforementioned chemical ingredients are shown in TM UFLC system . At a column temperature at 40 °C, substances were analyzed on an Atlantis T3  column. The mobile phase system was water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B). A flow rate of 0.4 mL/min and sample injection volume of 5 μL were used for gradient elution. The gradient elution program was established as follows: 0.01–0.3 min, 5% B; 0.3–0.8 min, 5–50% B; 0.8–1.5 min, 50–70% B; 1.5–2.8 min, 70–85% B; 2.8–5.0 min, 85% B; 5.0–5.01 min, 85–5% B; and 5.01–7.0 min, 5% B.The liquid chromatographic separation process was operated on an XR LC-20AD ProminenceMass spectrometry quantitative analysis was conducted on a Q TRAP 4000 MS/MS system to collect data, and then analyst software  was used to handle data. In the MRM mode and taking nitrogen as the circulating gas, electrospray positive ionization (ESI+) was used to analyze four substances. The detailed MS parameters were established as follows: gas 1, 50 psi; gas 2, 40 psi; air curtain gas, 40 psi; ion spray voltage, 5500 V; ion source temperature, 500 °C. Declustering potential (DP), entrance potential (EP), collision energy (CE), and cell exit potential (CXP) were performed as parameters optimized for substances in the MRM mode. The metabolic samples were operated on an Agilent 1260 Infinity HPLC system. The gradient elution program was established as follows: 0.01–2.0 min, 5% B; 2.0–2.5 min, 5–50% B; 2.5–3.0 min, 50–70% B; 3.0–8.0 min, 70–75% B; 8.0–10.0 min, 75–85% B; 10.0–30.0 min, 85% B; 30.0–30.01 min, 85–5% B and 30.01–35.0 min, 5% B. Other liquid phase conditions were the same as those under TM ion source to collect data. Then, the Metabolite Pilot software  was used to handle data. In the positive and negative mode, nitrogen was used as the circulating gas for analysis. The detailed MS parameters were established as follows: gas 1, 50 psi; gas 2, 50 psi; air curtain gas, 30 psi; ion spray voltage, 5500 V/−4500 V; ion source temperature, 550 °C; TOF MS scan, m/z 100–2000 Da; TOF MS DP, 80 V/−80 V; TOF MS CE, 10 V/−10 V; TOF MS/MS scan, m/z 50–1200 Da; TOF MS/MS DP, 80 V/−80 V; TOF MS/MS CE, 30 V/−30 V.Mass spectrometry qualitative analysis was conducted on a Q Triple TOF 5600 triple-time-of-flight hybrid mass spectrometer system with a DuoSprayAccurately weighed β-CD, DM, and internal standards reference substances were prepared as stock solutions, respectively, with concentrations of 1000 μg/mL, 500 μg/mL, and 500 μg/mL with methanol. Then, the stock solutions were serially diluted with methanol to achieve standard working solutions of needed concentrations: DM was 0.25–1000 ng/mL and β-CD was 0.05–100 µg/mL. Standard solutions were prepared by adding the working solutions into blank rat samples; 10 µL of β-CD working solution was added into 100 µL of blank rat samples. In blank rat plasma, urine, and feces, β-CD was set at concentrations of 0.05, 0.2, 0.5, 2, 5, 7.5, 10 µg/mL as linear samples and 0.15, 2.5, 8.0 µg/mL as quality control (QC) samples. In tissue homogenate, β-CD was set at concentrations of 0.005, 0.02, 0.05, 0.2, 0.5, 2, 5 µg/mL as linear samples and 0.015, 0.25, 4.0 µg/mL as QC samples. In blank rat plasma, tissue homogenate urine and feces, DM was set at concentrations of 0.025, 0.1, 0.5, 2, 10, 50, 100 ng/mL as linear samples and 0.075, 2.5, 80 ng/mL as QC samples. All added internal standards concentration were 0.5 µg/mL. The lowest concentration of the linear sample was LLOQ.  All sample solutions were kept at 4 ℃ before applying.v/v) was added, vortexed for 3 min, sonicated for 2 min in an ice-water bath, and centrifuged for 8 min at 16,000 rpm to deposit protein. The supernatant was transferred to a 1.5 mL Eppendorf tube, desiccated under airflow at room temperature, re-dissolved in 50 μL methanol, vortexed for 3 min, sonicated for 5 min in an ice-water bath, and centrifuged for 8 min at 16,000 rpm for LC-MS/MS analysis. For tissue and fecal samples, 100 mg of each sample was precisely weighed and added to a 5 mL Eppendorf tube, then mixed with 1 mL normal saline. The tissue cells were destructed by homogenization to mix with normal saline sufficiently, and the homogenization was achieved after centrifugation for 10 min at 4000 rpm. The methods to precipitate the protein of tissue and feces homogenization were the same as those of the plasma samples.The plasma and urine samples were kept at indoor temperature and dissolved before analysis. First, 100 µL of sample was added to a 1.5 mL Eppendorf tube together with 10 µL of ISs solution. Then, 300 µL of methanol:acetonitrile  was added, vortexed for 3 min, sonicated for 2 min in an ice-water bath, and centrifuged for 8 min at 16,000 rpm to deposit protein. The supernatant transferred to a 5 mL Eppendorf tube, desiccated under airflow at room temperature, re-dissolved in 80 μL methanol, vortexed for 3 min, sonicated for 5 min in an ice-water bath, and centrifuged for 8 min at 16,000 rpm for UHPLC-Q-TOF/MS analysis.Six rat plasma, tissue homogenates, feces, and urine samples were mixed evenly separately. First, 1 mL of sample was added to a 5 mL Eppendorf tube. Then, 3 mL of methanol:acetonitrile  were obtained from Changsheng Biotechnology Co., Ltd. . The selected rats without specific-pathogen-free (SPF), lived in a SPF experimental animal environment, with temperature 18–26 °C and humidity 40–70%. Rats were given normal diet and drinking water. Rats were fasted for 12 h before blood collection in order to avoid some ingredients in food affecting the experimental results. All experimental routines followed the “Principles of Laboratory Animal Care”. The animal experiment was performed under the animal experiment guidelines of Shenyang Pharmaceutical University and approved by the Animal Ethics Committee. p < 0.05 was considered as a difference, and p < 0.01 was considered as a significant difference.Eighteen male Wistar rats were separated into three groups with six rats in every group based on different oral medicines, containing a β-CD-DM group (inclusion compounds of DM with β-CD), DM group (single DM), and β-CD group (single β-CD). Each rat was administered to orally, and the dosage of every group was computed by the equal transformation of human and rat surface area of the body by the clinical dosage ; therefoEighteen male Wistar rats were separated into three groups with six rats in every group based on different times after administration, containing group A , group B , and group C . All Wistar rats were fed β-CD in a single oral administration at a dosage of 393 mg/kg. At 30 min, 1 h, and 4 h after oral administration, the rats were sacrificed by cervical dislocation. The rats were dissected immediately after death, and the tissues including heart, liver, spleen, lung, and kidney were collected. First, the tissues were sheared into a few pieces with scissors, and the rudimentary blood was washed with normal saline. Then, the tissues were stored at −80 ℃. The tissue homogenization samples of the rats in each group were treated according to the contents of Six male Wistar rats (250 ± 20 g) were placed in metabolic cages one day before administration to adapt to the environment, and blank urine and fecal samples were collected. All Wistar rats were fed β-CD in a single oral administration at a dosage of 393 mg/kg, after which urine and feces were collected over the periods 0–12 h and 12–24 h. The urine and feces samples of the rats in each group were treated according to the contents in TM software  for analysis. The minimum peak width and minimum retention time peak width were set to 25 ppm and 6 scans, respectively. The retention time and molecular weight error range were set to 0.50 min and 5 ppm, respectively.Six male SD rats were fed β-CD at a dosage of 393 mg/kg in continuous oral administration for 7d. Blood samples (250 μL) were obtained from the orbital venous plexus at 0, 0.25, 0.5, 1, 3, 6, 8, 12, 24 h after administration on the seventh day. Then, the blood was centrifuged for 10 min at 4000 rpm to obtain separation of plasma, and the plasma was kept at −80 ℃. Urine and feces were collected after administration. The rats were sacrificed by cervical dislocation 24 h after taking blood. The rats were dissected immediately after death and the tissues including heart, liver, spleen, lung and kidney were collected. The collected plasma, urine, and feces samples of rats in each group were treated according to the contents in The approach was conducted under the united instruction of the FDA Industrial Bioanalytical Method Validation guidelines and the Chinese Pharmacopoeia 2020 edition guidelines, including specificity, linearity, precision, accuracy, extraction recovery, matrix effect, stability, and dilution reliability.Specificity was evaluated by the differences of typical chromatograms of blank samples, blank samples added with analyte, and actual samples after oral administration.Two calibration curves were constructed daily and analyzed for three consecutive days to evaluate linearity.Six replicates of LLOQ and six replicates of three concentrations of QC samples were prepared and analyzed for three consecutive days to assess intra-day and inter-day precision and accuracy.The recovery rate was determined by comparing the peak area ratio of analyte and IS in six repeats of three concentrations of QC samples. The internal standard normalized matrix effect factor  was used to evaluate the matrix effect.The low and high QC samples were stored at room temperature for 7 h, in the autosampler for 8 h, low temperature for a long time (−80 °C for one month), after three freeze–thaw cycles. Then, the analytes were analyzed to evaluate the stability.Dilution reliability was calculated by analyzing six repeats of 80 μg/mL samples (10 folds the maximum QC concentration) diluted to 8.0 μg/mL with rat urine and feces.v/v) was chosen as an extraction agent for DM and β-CD, the extraction recovery rate was 90%, the matrix effect was good, the specificity was strong, and the sensitivity was high. In the majority of liquid phase terms, the chromatographic column played an important role in the separation of various analytes. It had strong polarity and weak retention because of the polyhydroxy cyclic structure of β-CD. By contrast, DM possessed a feebleness polarity feature. On account of the enormous polarity differences between β-CD and DM, a chromatographic column that provides satisfactory retention nature for both strong and feebleness polarity substances was selected for simultaneously detecting the two compounds. Atlantis T3, which is a highly hydrophilic reversed-phase chromatography column, could be a reliable selection. Two analytes and internal standards were isolated in 7 min by uncomplicated gradient elution on the Atlantis T3 column with greater separation. Water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) were chosen as the mobile phases because of the resulting satisfactory chromatogram shapes and better separation effect. Ginsenoside Re (IS 1) was chosen as IS of β-CD, and psoralen (IS 2) was chosen as IS of DM due to its appropriate retention time and mass response compere to analytes.This approach aimed to achieve sensitive and accurate detection of the β-CD in rat plasma. Because of the properties of cyclic oligosaccharides, the structure of the open chain was occupied less often, and it principally resided as an invariable circle structure in the process of ionization . In thisThe retention times of the typical chromatograms of blank plasma, blank plasma spiked with β-CD, DM, ISs, and plasma samples after oral medication were assessed to determine the specificity of β-CD and DM. There was no obvious peak disturbance at the retention times of β-CD (2.40 min) and DM (3.28 min), and Ginsenoside Re (IS 1) (3.61 min) and psoralen (IS 2) (3.70 min), as shown in 2 = 0.9977) and from 0.025 to 100 ng/mL of DM (R2 = 0.9970). The linearity of calibration curves of β-CD and DM is shown in There was a good linearity from 0.05 to 10 µg/mL of β-CD (RIn order to assess the precision (RSD) and accuracy (RE) of the method, the RSD and RE for LLOQ and QCs at three concentration levels, repeated six times, were determined. The inter-day RSD ranged from 6.10–12.96%, and the intra-day RSD ranged from 2.12–7.76%. The RE ranged from 0.02–6.28%. The detailed accuracy and precision of β-CD and DM are shown in The extraction recovery of β-CD was greater than 81.22%, that of DM was greater than 85.51%, and that of the internal standards was greater than 91.01%, verifying the effectiveness of the extraction method quantitatively. The matrix effect of β-CD was 91.21–92.80% and DM was 88.74–114.20%, indicating that the endogenous matrix during sample processing did not affect the quantification of the analytes.The stability RSD of β-CD and DM was 0.51–5.53%. The stability results showed that the samples could still be accurately quantified after being stored under different conditions.Note that due to the high concentration of β-CD in rat urine and feces, which exceeded the set range of the standard curve, we need to ensure dilution reliability. The RE and RSD of dilute QC samples were within 15%, verifying that the dilution process had no effect on the quantification of β-CD. The higher concentration of samples that exceeded the range of linearity could be properly diluted to precisely measure the concentration of β-CD. To ensure the accuracy and applicability of tissue distribution research and excretion research results, rat blank mixed tissue homogenization , rat blank urine and feces homogenization were used as the matrix to method validation, similar to rat plasma. The above results demonstrated that our approach could exactly and repeatedly measure the concentration of β-CD and DM in rat samples.The average plasma concentrations over time in rats after single oral administration of β-CD and DM are shown in 0−∞, Cmax, t1/2, and CLZ. From 0−∞ and t1/2 of DM in inclusion compounds were significantly increased, and CLZ was decreased.The differences between inclusion compounds and single compounds were mainly reflected in the pharmacokinetic parameters AUC0–t accounted for more than 96% of AUC0–∞, demonstrating that β-CD was completely eliminated in rat plasma 36 h after administration.From the concentration–time plots of β-CD in 0–∞. There was covalent bond formation or breaking during the construction of the inclusion compounds. It took longer for guest and host molecules to be in a dynamic balance, with t1/2 significantly increased and CLZ significantly decreased [The guest molecule DM is poorly water-soluble and belongs to the low-solubility and high-permeability drugs of the II class in the biopharmaceutics classification system (BCS), and its dissolution is a rate-limiting process, so it is mainly sold in the form of its hydrobromide (HBr) salt form . In geneecreased .The result of tissue distribution are shown in The excretion results are shown in Cyclodextrin was considered as the carrier of active ingredients until paying attention to the possible complexation of essential components in the intestine or blood, and it is speculated that it has physiological effects and participates in the metabolism in vivo . Because2-R1 transforming into R-CO-R1, with a carboxyl (-COOH) group forming at the branched chain. In fecal samples, the oxidation reaction of β-CD in the branched-chain formed carboxyl (-COOH) as I metabolism, followed by taurine conjugation reaction as per II metabolism, which was R-COOH transforming into R-CONH-CH2CH2SO3H. Maltose amylase was generated in the cytoplasm and displayed higher hydrolytic competence to β-CD, and the products were mainly maltose and sugar molecules of various lengths [2 and H2O [In fact, β-CD has resistance to gastric acid or saliva and pancreatic amylase, and it is widely hydrolyzed in the colon. β-CD is a cyclic oligosaccharide with non-reducing properties consisting of glucose units connected by α-1,4-glycosidic bonds , and a r lengths . These p and H2O . Moreove2 and H2O. Our results showed that β-CD had relative low accumulation in the body and the excretion and metabolism of β-CD were relatively rapid in vivo. Our results will be helpful to further study the design and safety evaluation of adjuvant β-CD and promote its clinical development.In this research, a specific and accurate LC-MS/MS approach was developed for the quantitative measurement of dextromethorphan hydrobromide and β-CD. Simultaneous determination and pharmacokinetic study of active ingredients and excipient in pharmaceutical preparations were achieved first. To offer an integrative representation of the biological fate of β-CD, the pharmacokinetics, tissue distribution, excretion, and metabolism of excipient β-CD were studied. The results showed that the effect of inclusion compounds changed the biological process, elimination rate, and exposure amount of β-CD and dextromethorphan hydrobromide in vivo. The study showed that β-CD was almost wholly removed from rat plasma within 36 h, and high concentrations of β-CD existed in organs with a high perfusion rate. A large proportion of β-CD was excluded from urine and feces, and it might be metabolized to maltodextrin. Sugar molecules with various lengths were further metabolized, absorbed, and finally expelled in the form of CO


Daily quantitative MR imaging during radiotherapy of cancer patients has become feasible with MRI systems integrated with linear accelerators (MR-linacs). Quantitative images could be used for treatment response monitoring. With intravoxel incoherent motion (IVIM) MRI, it is possible to acquire perfusion information without the use of contrast agents. In this multicenter study, daily IVIM measurements were performed in prostate cancer patients to identify changes that potentially reflect response to treatment.Forty-three patients were included, treated with 20 fractions of 3 Gy on a 1.5 T MR-linac. IVIM measurements were performed on each treatment day. The diffusion coefficient (D), perfusion fraction (f), and pseudo-diffusion coefficient (D*) were calculated based on the median signal intensities in the non-cancerous prostate and the tumor. Repeatability coefficients (RCs) were determined based on the first two treatment fractions. Separate linear mixed-effects models were constructed for the three IVIM parameters.−3 mm2/s, 0.086, and 28.7 × 10−3 mm2/s in the non-cancerous prostate and 1.19 × 10−3 mm2/s, 0.088, and 28.9 × 10−3 mm2/s in the tumor, for D, f, and D*, respectively. The repeatability coefficients for D, f, and D* in the non-cancerous prostate were 0.09 × 10−3 mm2/s, 0.05, and 15.3 × 10−3 mm2/s. In the tumor, these values were 0.44 × 10−3 mm2/s, 0.16, and 76.4 × 10−3 mm2/s. The mixed effects analysis showed an increase in D of the tumors over the course of treatment, while remaining stable in the non-cancerous prostate. The f and D* increased in both the non-cancerous prostate and tumor.In total, 726 fractions were analyzed. Pre-treatment average values, measured on the first fraction before irradiation, were 1.46 × 10It is feasible to perform daily IVIM measurements on an MR-linac system. Although the repeatability coefficients were high, changes in IVIM perfusion parameters were measured on a group level, indicating that IVIM has potential for measuring treatment response. Integrated MR-linac systems combine an MRI scanner with a linear accelerator, allowing acquisition of MRI scans of the patient on each treatment fraction of a radiotherapy (RT) course. On two commercially available systems, acquisition of quantitative MRI was shown to be feasible , 2. DailQuantitative MRI enables the characterization of tissue properties in a quantitative manner. By measuring this on a daily basis, two exciting ideas for personalized radiotherapy come within reach. The first is to adapt the dose distribution of a treatment plan on a daily basis according to the changing patient biology , and thePerfusion is of interest as it is related to tumor hypoxia, which is a prognostic marker for overall survival in a number of tumor sites . An estaChanges in IVIM parameters during treatment might provide valuable information about treatment response . For cerForty-three patients from three institutes with intermediate- and high-risk biopsy-proven prostate cancer were included in this study according to the EAU risk classification . Twelve 2-weighted scan for position verification and an IVIM scan were acquired before the start of irradiation. Thus, the scans on the day of the first fraction provide pre-treatment information. All institutes used the same protocol for the IVIM scan. For the development of the IVIM protocol, previously published guidelines were followed for ADC measurements on the Unity MR-linac  under the assumption that the contribution of perfusion to the signal at these b-values is negligible Finally, D* was calculated using the obtained values of D and f in combination with the signal intensity at the lowest two b-values  of each IVIM parameter was calculated using variance , 26. Thep-value was calculated using an F-test based on Satterthwaite’s approximation.To analyze the evolution over time, linear mixed effects analysis was performed using R (v3.6.1) and the lme4 package . SeparatFor logistical and technical reasons, IVIM scans were missing in 56 out of the total of 860 fractions. Of the remaining 804 fractions, 73 were excluded because of anatomical deformations or susceptibility distortions caused by the EPI readout. Five were excluded because the patient moved between the acquisition of different b-values. This left 726 fractions for analysis with a median number of 18  available fractions per patient. In four patients, a tumor could not be distinguished and was not delineated. For those patients, the entire prostate region was analyzed as non-cancerous. −3 mm2/s in the non-cancerous prostate, which was significantly higher (p < 0.001) than in the tumor (1.19 ± 0.04 × 10−3 mm2/s). The pre-treatment average and standard error of the mean of f were 0.086 ± 0.005 in the non-cancerous prostate and 0.088 ± 0.01 in the tumor. The pre-treatment average and standard error of the mean of D* were 28.7 ± 1.4 × 10−3 mm2/s in the non-cancerous prostate and 28.9 ± 5.4 × 10−3 mm2/s in the tumor. The pre-treatment values of f and D* were not significantly different between the non-cancerous prostate and tumor.IVIM scans were available for the first fraction in 35 patients. The pre-treatment average and standard error of the mean of D were 1.46 ± 0.02 × 10−3 mm2/s for D, 0.05 for f, and 15.3 × 10−3 mm2/s for D*. In the tumor, the RCs were 0.44 × 10−3 mm2/s, 0.16, and 76.4 × 10−3 mm2/s for D, f, and D*, respectively. 3, whereas the median volume of the tumor delineations was 1.0  cm3. As shown in 3, and 3.The RC in the non-cancerous prostate was 0.09 × 10p-value = 0.003), the ROI , the fraction  and interaction terms between ISUP group and fraction  and between ROI and fraction . −3 mm2/s. The D in the non-cancerous prostate was 0.24 ± 0.03 × 10−3 mm2/s higher than in the tumor. Both ISUP groups and ROI had an interaction term with the fraction number, meaning that the change in D over the course of treatment was different for these groups. In the tumor, for patients with a low ISUP score, the tumor D increased 0.005 ± 0.001 × 10−3 mm2/s/fraction, whereas for the group with a high ISUP score, the increase was 0.007 ± 0.001 × 10−3 mm2/s/fraction. This reduces the difference in the D between these groups over the course of treatment: at the 20th fraction, the D as estimated from the model in the tumors of the low ISUP group is increased to 1.38 ± 0.02 × 10−3 mm2/s and that in the high ISUP group is increased to 1.33 ± 0.03 × 10−3 mm2/s.To analyze the evolution over time, mixed effects models were constructed for each IVIM parameter. The set of fixed effects and regression coefficients for each parameter are listed in p-value = 0.03), the fraction , and the interaction between fraction and institute . As institute is part of the interaction term, it was also added to the model as a fixed effect   at 0.001 ± 0.0005 per treatment fraction, which was the only significant effect containing institute. For the perfusion fraction f, the significant fixed effects were ROI  and ADT   is higher than previously reported (range 0.13–1.06 × 10−3 mm2/s)  . The low3 mm2/s) .−3 mm2/s between tumor and non-cancerous prostate reported by He et al.  Biomedical Research Centre at The Royal Marsden NHS Foundation Trust and the Institute of Cancer Research, London. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. The University of Tübingen receives funding from the German Research Council (DFG) grant nos. ZI 736/2-1 and PAK 997/1: MU 4603/1-1 and OT 534/3-1.The Netherlands Cancer Institute, the Institute of Cancer Research and Royal Marsden NHS Foundation Trust, and the University of Tübingen are members of the Elekta MR-linac consortium, which aims to coordinate international collaborative research relating to the Elekta Unity (MR-linac). Elekta and Philips are commercial partners within the consortium. Elekta financially supports consortium member institutions with research funding and travel costs for consortium meetings. AT declares research funding from Elekta, Varian, and Accuray. DT declares institutional collaborations including financial and non-financial support with Elekta, Philips, Dr Sennewald, PTW Freiburg, and TheraPanacea. The Department of Radiation Oncology Tübingen (DT and AM) has research collaborations with Elekta AB (Sweden), Philips (The Netherlands), and Siemens Healthineers (Germany).The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TOTAL) showed excellent reliability . Moreover, FGBTOTAL was strongly correlated with aerobic fitness , maximal workload , time to gas exchange threshold , and peak oxygen uptake . We also found that agreement between standardized FGB and standardized aerobic performance indices such as Texh, VO2peak, Wmax, maximum heart rate, TGET, and workload at gas exchange threshold was high by the Bland–Altman method. In conclusion, FGB is a reliable test that can be used in order to measure changes in cross-training performance caused by an intervention. Moreover, FGB is strongly correlated to aerobic fitness.Cross-training is a high-intensity functional training (HIFT) with multiple workout modalities. Despite the increasing number of studies in HIFT, there is still no validated test to measure its specific performance. It would also be advisable to determine whether selected cross-training workouts can implement a stimulus corresponding to maximize aerobic work. For these reasons, the purpose of our study was to evaluate the repeatability and reliability of Fight Gone Bad (FGB) workout and to assess its relationship with aerobic fitness. Twenty-one cross-training participants  finished the study protocol which included three two-day measurement sessions separated by 10 days. During each session, participants had their body composition measured, and they performed two exercise tests. The first test was an incremental cycling test to measure aerobic fitness, and the second was a cross-training specific FGB workout performed the next day. Reliability and repeatability were calculated from the three measurements. The total FGB Score (FGB Cross-training can be described as high-intensity functional training (HIFT) that incorporates exercises with barbells, kettlebells, dumbbells, cardiovascular tasks , and gymnastics 4. It is varied in its nature, which appeals strongly to many people around the world.Cross-training has incredibly grown in popularity for over a dozen years. It combines ten key physical parameters to improve overall fitness: cardiovascular/respiratory endurance, stamina, strength, flexibility, power, speed, coordination, agility, balance, and accuracy5 suggests that HIFT increases aerobic fitness, strength, musculature, and endurance. Cosgrove et al.6 studied the influence of six months of HIFT on multiple fitness characteristics. The participants completed three separate days of assessments across 10 fitness domains before and after participating in the program for 6 months. They found that HIFT improved several fitness parameters, including flexibility, power, muscular endurance, and strength6. Murawska-Cialowicz et al.7 also showed that there was an improvement in aerobic fitness after 3 months of HIFT training in women but not in men. However, no changes in Wingate anaerobic power were observed.In a systematic review, Haddock et al.11. Butcher et al.9 studied whether physiological measures can predict selected cross-training benchmark performance. Aerobic fitness was measured as maximum oxygen uptake (VO2max) in a treadmill ramp test9. Anaerobic power was measured in a classical Wingate test on a cycloergometer. Cross-training performance was evaluated in benchmark workouts; ‘Fran’ (three rounds of thrusters (a thruster is a combination of a front squat and an overhead press) and pull-ups for 21, 15, and 9 repetitions), ‘Cindy’ , ‘Grace’ (30 clean and jerks for time) and ‘CrossFit Total’ (1 repetition maximum (1RM) back squat, overhead press, and deadlift). They found no correlation between HIFT performance and aerobic fitness. Bellar et al.8 aimed to investigate the relationship of aerobic fitness and anaerobic power with performance in two representative cross-training workouts. The first was 12 min , and the second was based on the total time to complete the prescribed exercise  in cross-training experienced and naïve adults8. The cross-training experienced group performed significantly better in both performance tests. In the experienced group, both cross-training tests results were correlated with VO2max, but in the naïve group, only the 21–15–9 test. Dexheimer et al.10 conducted a study aimed to determine which physiological performance measure could serve as the greatest indicator of cross-training workout performance. Participants completed a graded exercise test on a treadmill to determine VO2max, a 3-min all-out test, a Wingate test, ‘CrossFit Total’, and three HIFT benchmark trainings: ‘Grace’, ‘Fran’, and ‘Nancy’ (5 rounds of a 400 m run and 15 overhead squats). In comparison to Butcher and colleagues9, they found that ‘Fran’, ‘Nancy’, and ‘CrossFit Total’ were significantly correlated with VO2max, but only the score in ‘Nancy’ and ‘CrossFit Total’ were correlated with peak and average anaerobic power10. Interestingly, they showed that critical speed and total body strength are not crucial predictors of HIFT performance. Finally, Martínez-Gómez et al.11 hypothesized that a full squat might be used as a predictor of cross-training performance, at least for exercises involving lower-limb muscles. The participants performed a squat test to measure 1RM and five cross-training workouts from the CrossFit Opens in 2019. In the first workout of the day (WOD1), participants had to perform 225 repetitions of dumbbell snatches and burpee box jump-overs for time. In WOD2, the athletes had to perform as many repetitions as possible of 50-feet (15.24 m) weighted walking lunges, toes-to-bar, bar muscle ups, and power cleans in 12 min. In WOD3, contestants had to perform the maximum possible number of repeated circuits, each including chest-to-bar pull-ups and squat snatches (with weight progressively increasing from 95 to 265 lbs (43–120 kg)) in 8 min. In WOD4, participants had to perform as many repetitions as possible of deadlifts (225 lb (102 kg)), wall-ball shots (20 lb (9 kg) to a 10-feet (3 m) high target), rowing, and handstand push-ups in 13 min. In WOD5, participants had to perform 440 repetitions of thrusters (95 lb (45 kg)) and double-unders in the shortest time possible. Cross-training performance was then calculated from all five WODs. They found moderate to strong correlations between squat variables and all WODs11. The same for cross-training performance.Other investigators focused on finding the connection between HIFT performance and aerobic and anaerobic fitness as well as strength capabilities13. However, it is difficult to compare those results, because authors use different cross-training tests. The reason for this is that there are no validated and well-described tests of cross-training performance. In the present paper, we proposed one of the cross-training benchmark workouts Fight Gone Bad (FGB) as a discipline-specific test to measure HIFT performance. FGB comprises 3 rounds of 5 different cross-training specific exercises, so there are 15 min of exercises with two 1-min breaks between the 1st and 2nd, and 2nd, and 3rd rounds. It requires most of the HIFT key physical traits such as power, strength, speed/strength endurance, balance, and stamina2. In addition, it can be performed by both experienced and inexperienced athletes, which makes it useful to track progress over time. That is why the purpose of this study is to evaluate the repeatability and reliability of FGB over repeated measurements and to assess its relation to aerobic performance. The secondary aim was to assess changes in hematological and biochemical parameters caused by FGB in order to have better insight into the organism’s response to such a workout.Because HIFT is becoming more and more popular every year, many studies are carried out to measure the influence of different dietary regimens and supplementation on cross-training performanceThere were no significant differences in body mass and composition between the three measurements (baseline and after 10 and 20 days), Table R-1) and FGBTOTAL and good for FGB Round-2 (FGBR-2) and FGB Round-3 (FGBR-3). SEM was 6, 8, 9 and 6% for FGBR-1, FGBR-2, FGBR-3, and FGBTOTAL, respectively. SEM was lower than SWC for FGBR-1 and FGBTOTAL, indicating good ability of the test to detect small and meaningful changes. However, for FGBR-2 and FGBR-3, SEM was higher than SWC. MDC values are given in Table There were no significant differences in FGB performance between the three measurements Table . ICC wasR-2, Rest2, Mean15min, and Mean17min. ICC was acceptable for Rest1 and FGBR-3 and low for FGBR-1. SEMs were low, between 2 and 5%. SEM was higher than SWC for all HR measurements in FGB. MDC values are given in Table There were no significant differences in HR during FGB between the three measurements Table . ICC wasexh, Wmax, TGET, WGET, VO2peak, VCO2peak, and EE, good for HRGET and VO2GET, and acceptable for HRmax. SEM was low (< 5.99%) for HRmax, HRGET, and VO2peak, and moderate (6–10%) for Texh, Wmax, TGET, WGET, and VO2GET. SEM for EE was 11%. SEM < SWC was observed for Texh, Wmax, TGET, WGET, VO2peak, VCO2peak, and EE. However, for HRmax, HRGET, and VO2GET SEM was higher than SWC.Furthermore, there were no significant differences in ICT Table . RelativGET  correlations were found for FGBR-1 with WGET, VO2GET, VO2peak, and VCO2peak, for FGBR-2 with Texh, Wmax, TGET, WGET, VO2peak, VCO2peak, and EE, for FGBR-3 with Texh, Wmax, TGET, WGET, VO2peak, and EE, and for FGBTOTAL with Wmax, TGET, WGET, VO2GET, VO2peak, and VCO2peak. In addition, correlations were significant but low for FGBR-1 with HRmax, FGBR-2 with HRmax, and VO2GET, FGBR-3 with HRmax, HRGET, VO2GET, and VCO2peak, as well as FGBTOTAL with HRmax.Correlations between FGB scores and ICT parameters were significant for all measurements besides HRET Table . Strong TOTAL and performance in ICT measured as standardized Texh, VO2peak, Wmax, and HRmax were 0.0 ± 0.70, 0.0 ± 1.53, 0.0 ± 0.74 and 0.0 ± 0.10, respectively  of blood parameters between FGBPRE, FGBPOST, ICTPRE, and ICTPOST. We found that WBC, LYM, MON, La, and Pa were significantly higher FGBPOST than FGBPRE and ICTPOST than ICTPRE  were higher than SEM for FGBR-1 and FGBTOTAL, indicating the ability of test to detect small and meaningful performance changes. Interestingly, the repeatability of body composition measurements in our study indicates that the participant did not implement any changes in their lifestyles throughout the study , that could influence the performance.In practical and scientific respects, the reproducibility of a test is essential to determine whether an individual has experienced a training response. Moreover, reliability estimates the extent to which the change in the measured score is due to a change in the true scoreexh, Wmax, TGET, WGET, VO2peak, VCO2peak and EE), ICC was close to 1.0 and SEM was low. This is in accordance with previous studies. Dideriksen and Mikkelsen17 showed excellent ICC (< 0.9) for VO2max, Wmax, and HRmax and good ICC (0.7–0.9) for VO2VT in recreationally trained triathletes (n = 13). Weston and Gabbett18 found ICC > 0.9 for VO2max, VE, VCO2, HR, and W, with measurement errors below 5% in trained cyclists (n = 16). Graded exercise testing is a reliable tool that is widely used for the determination of VO2peak in sports performance, research, and clinical diagnostics19. However, this is beyond the scope of the present paper.Moreover, relative reliability was even better for ICT, where for most of the measured parameters . One explanation, though not strong enough, for such a phenomenon can be that the duration of FGB forces the engagement of the aerobic energy system. The other reason for that can be in the main characteristic of FGB, which is the 1-min rest periods between the rounds. It seems that the ability to recover between bouts of exercise is dependent upon oxidative capacity20. One study showed that better recovery between repeated bouts of Wingate sprints was associated with better cross-training performance20. Therefore, it suggests that even if rounds in FGB are to some extent anaerobic in nature, the ability to sustain effort throughout the entire FGB may be reliant on the aerobic recovery efficiency during breaks between the rounds. This is in accordance with literature suggesting that aerobic capacity enhances recovery from high intensity intermittent exercise through increased aerobic response, improved lactate removal, and enhanced phosphocreatine regeneration21.Considering speed and strength efforts, Fight Gone Bad (FGB) is a high-intensity workout of moderate duration . It is performed very fast and demands a high level of muscular endurance. The present study compared the results in FGB to aerobic fitness measured in incremental cycling test. We found strong and moderate correlations between FGB performance and time to exhaustion, maximum workload, VO8 found that cross-training performance was correlated to aerobic power (VO2max) but only in experienced athletes  and not in the naïve . The cross-training workout they used consisted of 21–15-9 repetitions of (1) sumo deadlift high pull, (2) box jump (50 cm), and (3) 40-m farmer's walk gripping two 20-kg bumper plates. The score was the time for workout completion. In yet another paper, Dexheimer et al.10 showed that the higher the VO2max the better the result in ‘Fran’, ‘Nancy’, and ‘CrossFit Total’. However, in the regression model, VO2max explained 68% of the variance only in ‘Nancy’10. ‘Fran’ consisted of 3 rounds of 21–15-9 repetitions of thrusters and pull-ups. ‘Nancy’ was a workout of 5 rounds of a 400 m run and 15 overhead squats with a barbell (95 lb men/65 lb women). ‘CrossFit Total’ included 1-repetition maximum (RM) back squat, strict shoulder-press, and deadlift. Interestingly, they did not find any significant association between VO2max and the ‘Grace’ workout (30 clean and jerks (135 lb men/95 lb women)). The authors thus hypothesized that those with higher VO2max may perform better in longer workouts that require running  compared to shorter . In contrast to Dexhaimer, Butcher et al.9 found no correlation between VO2max and ‘Fran’, ‘Grace’, or ‘Cindy’  or ‘CrossFit Total’. However, they found significant correlations between VO2 at anaerobic threshold and ‘Fran’, ‘Grace’, and ‘CrossFit Total’. Furthermore, Martinez-Gomez et al.11 aimed to determine which physiological variables could predict performance during a cross-training competition . They found that the combination of lower-body muscle power (squat jump performance), reactive strength (reactive strength index during a drop jump), and aerobic power (as measured with the VO2max) together explained 81% of the cross-training performance variance, showing that HIFT performance is associated with a variety of fitness markers related to both aerobic and anaerobic/power capabilities. This seems to confirm that HIFT demands high adaptation to both aerobic and anaerobic types of exercise. Therefore, the improvement in cardiorespiratory fitness may enhance the performance in longer workouts like FGB or ‘Nancy’. Thus, it would be reasonable to include aerobic training in cross-training programming. Moreover, it would be also beneficial to include training like FGB in sports using varying energy systems like combat and team sports. In these sports, all three energy systems are used according to the intensity, rhythm, and duration of the competition. That is why they can benefit the most from FGB, which is of high intensity yet strongly correlated with aerobic fitness .Some investigators aimed to compare different physiological variables with HIFT performance. Bellar et al.POST and ICTPOST compared to FGBPRE and ICTPRE, respectively, but there were no differences in La or Pa between FGBPOST  and ICTPOST . GLU concentration increased significantly only FGBPOST versus FGBPRE (153.0 ± 44.6 mg/dL to 117.2 ± 22.3 mg/dL). In a previous study, Feito et al.20 observed similar concentrations of La in the last round of 15-min cross-training AMRAP . The 15-min AMRAP circuit consisted of 250-m of rowing, 20 Kettle bell swings (16 kg for men and 12 kg for women), and 15 dumbbell thrusters (16 kg for men and 9 kg for women). In another study, La and GLU concentrations were significantly different between WOD1 and WOD2 22. WOD1 (AMRAP) consisted of as many rounds as possible of burpees and toes to bar increasing repetitions  in five minutes. WOD2 was the number rounds for time (RFT) consisting of three rounds of 20 repetitions of wall ball (9 kg) and 20 repetitions of power clean (a load of 40% 1RM) in the shortest possible time. HR measurement showed that during WOD1 the majority of time was spent in the intensity zone of 50–59% HRmax, whereas during WOD2 it was in the zone of 90–100% HRmax. Thus, this explains the difference in La and GLU concentration. Fernandez-Fernandez et al.23 observed that La concentration increased from 4.0 ± 1.3 mmol/L to 14.5 ± 3.2 mmol/L in ‘Cindy’ and from 4.0 ± 1.3 mmol/L to 14.0 ± 3.3 mmol/L in ‘Fran’. Moreover, Tibana et al.24 observed lower concentrations of La after two different cross-training workouts, 11.84 ± 1.34 mmol/L after WOD1 and 9.05 ± 2.56 mmol/L after WOD2. WOD1 was 10 min of AMRAP of 30 double-unders and 15 power snatches (34 kg)24. WOD2 was 12 min AMRAP of rowing 250 m and 25 target burpees. They also observed an increase in GLU concentration (81.59 ± 10.27 mg/dL to 114.99 ± 12.52 mg/dL in WOD1 vs. 69.47 ± 6.97 mg/dL to 89.95 ± 19.26 mg/dL in WOD2).Also, the present study is the first to provide extensive data on biochemical response after each exercise test among HIFT-trained participants. La and Pa concentrations significantly increased FGB22 noted a significant increase in CK activity after two WODs (from 566.4 ± 159.1 IU/L to 689.6 ± 281.9 IU/L in WOD1 (5 min AMRAP) and from 406.8 ± 201.0 IU/L to 492.2 ± 203.8 IU/L in WOD2 (RFT)). CK remained elevated 24 h post exercise (864.0 ± 369.5 IU/L after WOD1 and 673.8 ± 444.1 IU/L after WOD2) and returned to baseline 48 h post exercise. However, they did not note any differences in LDH.In the present study, we did not observe any significant differences in CK and LDH activities. Timon et al.25. For this reason, we standardized post exercise biochemical and hematological parameters in relation to the hematocrit value. We observed significant differences in HGB and MON between T1, T2 and T3, yet they were not clinically important. WBC, LYM, and MON concentration increased after exercise (both FGB and ICT). GRA increased significantly only after ICT. Interestingly, LYM count was higher FGBPOST than ICTPOST, but pre-exercise values were also higher before FGB than before ICT. The increase in WBC can be attributed to increased blood flow that recruits the leukocytes from the marginal pool and/or hormonal changes which are likely to be mediated by beta-2 adrenergic receptors25. In contrast to our study, in sedentary adults acute high-intensity interval training (HIIT) increased HGB concentration from 15.75 ± 0.76 g/dL pre-exercise to 16.59 ± 0.81 g/dL post-exercise and RBC count from 5.44 ± 0.22 × 1012/L to 5.92 ± 0.22 × 1012/L26. In addition, they observed a significant increase in WBC (from 7.32 ± 1.83 × 109/L to 12.84 ± 3.37 × 109/L) and LYM (3.11 ± 1.59 × 109/L to 5.22 ± 1.99 × 109/L) but not in MON. The differences between individual studies most often result from not using the hematocrit conversion formula that takes into account post-exercise dehydration and the transfer of fluids from the bloodstream to the tissues27. This can explain the differences in blood cell counts observed by Belviranli et al.26.Finally, it has been generally shown that acute exercise increases erythrocytes, leucocytes and platelet counts, hematocrit values, and hemoglobin concentration significantly as compared to pre-exercise values, and these increments depend on fluid shifts (plasma volume contraction) caused by the exercisemax measured in ICT, we could assume that FGB was very intense and that every time the participants put in a lot of effort. We also controlled the biochemical and hematological parameters throughout the study, which gave us insight into metabolic changes in relation to both exercise tests.Our study is the first to evaluate the repeatability of a cross-training test. The strength of our study is that the participants performed FGB and ICT three times in the same conditions, which allowed more precise assessment of reproducibility. Moreover, we controlled whether the participants implemented any significant changes in their lifestyles, i.e., reduction diet, by measuring body composition. We also controlled the HR during the test to evaluate the intensity of exercise. By comparing average FGB HR to HRThe biggest limitation of our study is that we only measured the correlation of FGB with aerobic fitness. HIFT is varied in its nature, because it combines strength, power, speed, agility, and cardiovascular fitness. For this reason, we claim that future studies should evaluate the relationship between FGB performance and other physiological parameters such as anaerobic power or strength. Even though our study shows that FGB gives reliable scores, it seems important to evaluate its connection with other physical traits. It should also be taken into account that we used a cycloergometer for aerobic fitness evaluation in this study, which, to some extent, could affect the obtained aerobic fitness results. It is well known that aerobic power measured on a treadmill is higher than on a cycloergometer, because running engages whole-body and cycling mostly lower-body movements. However, the choice of a cycloergometer was motivated by the reluctance of our study group towards running. Thus, there was a justified concern that the participants would not fully engage in a graded running test. It is also worth-noting that the participants were well adapted to perform lower-body movements , which could be beneficial for cycling performance.Our study showed that Fight Gone Bad is a reliable and repeatable test to measure cross-training performance. Moreover, FGB is strongly correlated with aerobic fitness. FGB can be used as a tool in interventional studies to evaluate the changes in cross-training scores. Furthermore, given that FGB is a non-invasive, easy to perform, and accessible test, it can be regularly used by coaches throughout the training season.2max 3191 ± 823 mL/min) completed the entire study protocol and were included in the analyses  58.7 ± 13.9 kg, fat mass (FM) 19.1 ± 7.1%, and VOThe study protocol was reviewed and approved by the local ethical committee . Each subject was informed of the testing procedure, its purpose, and the risks of the study. Each participant submitted her/his written consent to participate. All procedures were conducted in accordance with the ethical standards of the 1975 Helsinki Declaration.1), and after 10 (T2) and 20 (T3) days, respectively  at the Poznan University of Life Sciences and selected “Cross-training Boxes” in Poznan at baseline  based on the previously mentioned recommended procedures28.Body mass (kg) and height (cm) were measured using a professional medical scale with a stadiometer  at an accuracy of 0.1 cm and 0.1 kg for height and body mass, respectively. FM and FFM were assessed based on air displacement plethysmography using the Bod Pod  as described previously1, T2, and T3). Between ICT and FGB tests at least a 30-h recovery break was implemented. Prior to each tests (ICT and FGB), participants were given instructions on the procedure, and they completed a brief warm-up period  of approximately 50 W power and ~ 70 rpm cadence, followed by a 5-min light stretching and 5-min break). All tests were performed in proper workout clothing and shoes, and the tests were supervised by an experienced researcher. Heart rate was continuously monitored during exercise using a telemetric system . Furthermore, capillary blood samples were obtained for analysis before and after each test. During exercise, all test participants were verbally encouraged to maximize their efforts.The study protocol consisted of the incremental cycling test (ICT) and FGB workout performed 3 times (T2peak), and gas exchange threshold (GET). We considered the VO2peak to be the moment when the individual oxygen uptake (VO2) recorded during the ICT reached the highest point29. To determine the GET during the ICT, the V-slope method was applied based on an analysis of the linear regression for the curve of increasing CO2 exhalation in comparison to the curve of increasing O2 uptake32.The initial load was set at 50 W for women and 75 W for men and increased every 1.5 min by 25 W until volitional exhaustion. Respiratory parameters and heart rate (HR) were measured (breath by breath) by the Quark CPET ergospirometer . Measured variables included time to exhaustion (Texh), maximal workload (Wmax), maximum heart rate (HRmax), time to GET (TGET), workload at GET (WGET), heart rate at GET (HRGET), oxygen uptake at GET (VO2GET), VO2peak, peak carbon dioxide production (VCO2peak), and energy expenditure (EE).An exercise test on the Kettler X1 cycloergometer  was performed to determine peak oxygen uptake  before beginning the next round34. Wall balls combined a front squat with a medicine ball  and a push press-like throwing of the ball to a target located 2.75 m for females and 3.0 m for males. At the bottom of the squat, the hips should be lower than the knees. In sumo deadlift high pull, the feet were wider than the hips, and the grip was inside the knees. The exercise started with lifting the bar  from the ground like in classical deadlift, but then the bar was pulled to the chest. At the end, the elbows should be higher than the shoulders. The Box jump started with both feet on the ground. Athletes jumped on a box that was 50 cm tall for females and 65 cm for males with landing on both feet. The exercise ended when shoulders, hips, and knees were extended in one line. Push press started with lifting the bar  from the ground to the front rack. Then the bar was pushed overhead using leg power. After the shoulders were straight, the bar was dropped back to the shoulders. Rowing was performed on an ergometer. Feet were taped to the feet plates with special straps. The handle was pulled towards the chest, using the push from the knees. The test was video recorded in order to allow an accurate count of all properly done repetitions. For each valid repetition, a participant needed to complete a full range of motion required for a specific exercise.Fight Gone Bad comprised three rounds of five exercises: wall ball, sumo deadlift high pull, box jump, push press, and rowingPRE and FGBPRE) and 3 min after exercise tests (ICTPOST and FGBPOST) capillary blood was collected from a fingertip of the nondominant hand using a disposable lancet-spike Medlance Red  with a 1.5 mm blade and 2.0 mm penetration depth as described previously13. Approximately 300 μL of blood was collected into a Microvette CB 300 tube  containing K2-EDTA  as anticoagulant for hematological measurements. Blood sample tests were carried out with the use of an 18-parametric automated hematology analyzer Mythic 18 . The count of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocyte (GRA), red blood cells (RBC), platelets (PLT), as well hemoglobin (HGB) concentration and hematocrit (HTC) value were considered in the study. Furthermore, another 300 μL of capillary blood was collected in a Microvette CB 300 Z tube  with a clotting activator, in which the activities of creatine kinase  and lactate dehydrogenase  were measured using an optimized kinetic methods. In addition, 50 μl of capillary blood was collected into a neutral (without anticoagulant) glass capillary . The blood samples were deproteinized in 0.6 mol/L of perchloric acid (HClO4). After centrifuging (4000 g/10 min/4 °C), the supernatant was isolated. The enzymatic measurements of lactate (La) and pyruvate (Pa) concentrations were based on methodology proposed by Maughan35. Glucose concentration was detected with an enzymatic, colorimetric PZ Cormay test . All biochemical measurements were conducted using a multi-mode microplate reader . To avoid the influence on biochemical and hematological parameters caused by changes in plasma volume during physical effort, an appropriate hematocrit converter formula was used34.Blood was collected by qualified medical personnel in accordance with applicable procedures. Before  between T1, T2, and T3. The ICC gives the ratio of variances due to differences between subjects. ICC < 0.40 was considered low, between 0.40 and 0.70 acceptable, between 0.70 and 0.90 good, and > 0.90 excellent. However, ICC does not give an indication of the accuracy of individual measurements. Absolute relativity was calculated as standard error of measurement (SEM), which quantifies the precision of the individual measurements. The usefulness of the test was assessed by calculating the smallest worthwhile change (SWC). The ability of the test to detect small and meaningful changes was rated as good if SEM ≤ SWC, satisfactory when SEM = SWC, and marginal in cases with SEM ≥ SWC. Minimal Detectable Change (MDC), which is the minimal amount of change that a measurement must show to be greater than the within subject variability and measurement error, also referred to as the sensitivity to change, was also calculated. Associations between the FGB score and aerobic capacity were measured using the Pearson correlation coefficient. The following criteria were adopted for the interpretation of the magnitude of the correlation: trivial (r < 0.1), small (0.1 ≤ r < 0.3), moderate (0.3 ≤ r < 0.5), large (0.5 ≤ r < 0.7), very large (0.7 ≤ r < 0.9), nearly perfect (0.9 ≤ r < 1), and perfect (r = 1). The agreement of two methods was evaluated using the Bland–Altman method after data normalization36. Normalization was done by subtracting the mean and dividing by standard deviation.Normal distribution was examined using the Shapiro–Wilk test. Differences between TAll procedures performed were in accordance with the ethical standards of the institutional and national research committee ) and with the 1975 Helsinki declaration and its later amendments or comparable ethical standards.All participants signed an informed consent.This work proposes the first evaluation of the reliability and validation of a specific test to measure HIFT performance. Our study indicated that FGB is a reliable test that can be used in order to measure changes in cross-training performance caused by an intervention. We also showed that cross-training performance is correlated to aerobic fitness, which gives more insight into the physiology of the test. It shows that aerobic fitness, even though underestimated by most of the cross-training athletes, can be an important contributor to success. Our findings could serve as guidance for scientists, as well as coaches and athletes who consider achieving their scientific and/or training goals based on the cross-training specific Fight Gone Bad workout.