The Efficacy of Chlorella Supplementation on Multiple Indices of Cycling Performance

Abstract This study investigated the effects of chlorella supplementation on submaximal endurance, time trial performance, lactate threshold, and power indices during a repeated sprint performance test by fourteen male trained cyclists. Participants ingested 6 g/day of chlorella or placebo for 21-days in a double-blinded randomized counter-balanced cross-over design, with a fourteen-day washout period between trials. Each completed a 2-day testing period comprising a 1-hour submaximal endurance test at 55% external power output max and a 16.1 km time trial (Day-1), followed by a lactate threshold (Dmax) and repeated sprint performance tests (3 X 20 s sprints interspersed by 4-mins) (Day-2). Heart rate (b.min−1), RER, V̇O2 (ml·kg−1·min−1), lactate and glucose (mmol/L), time (secs), power output (W/kg), and hemoglobin (g/L) were compared across conditions. Following chlorella supplementation (chlorella vs. placebo for each measurement) average lactate and heart rate were significantly lower (p < 0.05) during submaximal endurance tests (1.68 ± 0.50 mmol/L vs. 1.91 ± 0.65 mmol/L & 138 ± 11b.min−1 vs. 144 ± 10b.min−1), average power and peak power (W/kg) were significantly higher during repeated sprint bouts (9.5 ± 0.7 W/kg vs. 9.0 ± 0.7 W/kg & 12.0 ± 1.2 W/kg vs. 11.4 ± 1.4 W/kg), hemoglobin significantly increased (149.1 ± 10.3 g/L) in comparison to placebo (143.4 ± 8.7 g/L) (p = 0.05). No differences existed between conditions for all oxygen consumption values, 16.1 km time trial measures and lactate threshold tests (p > 0.05). In conclusion, chlorella may pose as an additional supplement for cyclists to consider, particularly for those cyclists who want to improve their sprinting.


Introduction
Microalgae are an emerging functional food source that is gaining traction and popularity in biopharmaceutical, nutraceutical, and biotechnology industries.They are a diverse and complex species that comprise an abundant breadth of micronutrients (multiple vitamins, minerals, fatty acids, and amino acids) that can promote human health (Wells et al. 2017), and possibly performance (Gurney and Spendiff 2022).Spirulina and chlorella are currently the most popular microalgae supplements on the market which possess similar nutritional qualities (see Andrade and colleagues (Andrade et al. 2018) for an in-depth comparison) and are readily available in powdered, capsule, and pill form.Previously the focus has been on their clinical application and potential (Wells et al. 2017), though some researchers have attempted to bridge the gap between these positive clinical findings and the potential for microalgae as an ergogenic aid (Gurney and Spendiff 2022).However thus far, there is a clear disparity in research volume within the Sport and Exercise Nutrition field between spirulina and chlorella.Spirulina has received the most attention yet has produced some equivocal results (Gurney and Spendiff 2022).Despite the similar nutritional qualities between spirulina and chlorella, chlorella contains higher concentrations of iron, omega-3 fatty acids, vitamin A, riboflavin, iron, magnesium, niacin and zinc (Andrade et al. 2018).Therefore, it's worthy to investigate whether chlorella has analogous function and potential during exercise.
In clinical trials and meta-analyses chlorella supplementation has previously generated an improvement in immune function (increased secretion of salivary secretory immunoglobulin A) within healthy males and females (Otsuki et al. 2011;Chidley and Davison 2018), as well as cardiovascular risk factors and blood lipid profiles within both male and female healthy and clinical populations (hypertension, dyslipidemia, diabetes, and nonalcoholic fatty liver disease) (Fallah et al. 2018;Sherafati et al. 2022).Chlorella's potential vasodilatory properties have also previously been investigated where reductions in arterial stiffness following a 6 g/day 28-day supplementation protocol in both young males and middle-aged individuals were reported (Otsuki et al. 2013;2015).Suggestions that the carotenoids (lutein, β-Carotene and zeaxanthin), polyunsaturated fatty acids (PUFA), linoleic acid and water-soluble fibers found in chlorella can bind to digested fat, increase plasma scavenging of LDL-C (low density lipoprotein), and reduce absorption of sterols (cholesterol) from the intestine were some of the purported mechanisms of action.
From an exercise performance perspective, significant augmentations in peak oxygen uptake in nutritionally insufficient young men following a 6 g/day 28-day supplementation protocol has been reported (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017), with the proposed mechanisms of action being improvements in nutritional status, as well as the arginine content in chlorella potentiating the production of Nitric Oxide (NO) -a known signaler of vasodilation.The iron content in chlorella has previously been attributed to improving Hb in rats (Gao et al. 2019) and pregnant women (Nakano et al. 2010), as such further research is warranted to establish whether chlorella supplementation may also induce similar effects in trained men.In exercising rodents, the influences of chlorella supplementation on exercise capacity and lactate metabolism have demonstrated positive results, with the evidence indicating chlorella may increase the expression of Monocarboxylate-transporters (MCT) and increase maximal swimming times (Mizoguchi et al. 2011;Horii et al. 2017).MCTs play an important role in facilitating lactate transport, and thus metabolism (Brooks 2018).Yet it is currently unknown as to whether chlorella may induce similar positive lactate metabolism results in humans.Efficient lactate metabolism is especially important for cycling whereby it is common for races to comprise uphill sections and intermittent sprint finishes where the prerequisite is to produce high intensity effort with the ability to use brief recovery periods to buffer lactate and any deleterious byproducts associated with fatigue (Bell et al. 2017).Consequently, recent reviews have highlighted the importance of lactate for energy metabolism during both aerobic and anerobic exercise intensities (Brooks 2018;2020).Furthermore, during cycling races, producing a continuous high relative power output is a requisite for success (Kordi et al. 2020;2020) as it has been proposed that peak power (PP) is a determinant of performance (Dorel et al. 2005).
Considering similar microalgae such as spirulina has focused on key performance indicators in cyclists (Gurney et al. 2021), it's worthy to explore whether chlorella may possess similar potential during the same given exercise tests and supplementation/ dose period.Indeed, to the best of our knowledge, no study has previously investigated whether a 6 g/day 21-day chlorella supplementation period is also efficacious.Therefore, the aim of this study was to investigate whether 6 g/day supplementation of chlorella for 21-days can improve and influence: respiratory variables and lactate during submaximal endurance tests, Hb, 16.1 km time trials (TT), lactate thresholds (LT), repeated sprint performance tests (RSPT).It was hypothesized that chlorella supplementation would lower homeostatic disturbance during submaximal cycling which may lead to improvements in cycling performances.

Participants
Initially eighteen participants were recruited however four were lost or did not meet the inclusion criteria (See Consort diagram in supplementary files).Therefore, fourteen trained healthy male experienced cyclists (Mean ± SD; Age 37 ± 8 years, Stature 184 ± 5 cm, Mass 78 ± 8 kg, average weekly self-reported kilometers/hours cycled 201.1 ± 67.0 km/8.5 ± 2.5 hrs, V ̇O2max 50.74 ± 5.69 ml•kg −1 •min −1 ) completed the study.Participants were required to provide written informed consent, train at least 4-5 h per week and have a minimum of 2-years of cycling experience.The exclusion criteria were any cyclist that currently smoked or had a history of cardiovascular disease.The Faculty of Science, Engineering and Computing Ethics Committee at Kingston University London approved the study (1819.056.1) in accordance with the Declaration of Helsinki.The study followed the CONSORT and Good Clinical Practice guidelines.Considering the submaximal heart rate results (Effect size = 0.73, observed power =0.90) and power calculation from Gurney et al. (2021), a minimum sample size of eight was required to detect a medium effect (0.5) of chlorella supplementation in a priori power analysis using an alpha of .05 and a power of .90(G*Power® software, version 3.1.9.7; Universität Kiel, Kiel, Germany (Faul et al. 2007)).

Study design
The study design and methodology closely follow those methods utilized in previous work (Gurney et al. 2021).Participants were required to visit the laboratory on six separate occasions at the same time of day (following an overnight fast) in a double-blinded randomized counter-balanced cross-over design.Participants were told to continue their regular training regime, and to refrain from taking any additional nutritional supplements during the intervention.48 hrs prior to each visit, participants were instructed to refrain from exercise and to record a food diary, this was replicated prior to each subsequent visit -adherence was 100%.Baseline anthropometric measurements, a V ̇O2max test, and familiarization of the testing protocols (Day 1) were conducted during visit one.During visit two, participants were accustomed to the exact same testing protocols which occurred on Day 2 following supplementation.Test-retest reliability, expressed as Coefficient of Variation % (CV%), for each performance test are the same values reported in our previous study (Gurney et al. 2021).For hemoglobin analysis, the HemoCue (AB Hb 201+, Ängelholm, Sweden) CV% was 1.2%.

Visit 1
Baseline measurements and V ̇O2max .At the beginning of each visit, body mass (kg) and a 10 µL sample of capillary blood for hemoglobin (g/L) analysis were taken.Participants adjusted the Monark 894E Peak Bike to their preferred saddle height, which was recorded and replicated for subsequent tests.For the 16.1 km TT, participants used a TT bike (Boardman Bikes Ltd, London, UK) fixed on a Tacx Turbo Trainer (i-Genius T2020, Fisher Outdoor Leisure PLC UK) which was calibrated following the manufacturer's instructions prior to each trial.All tests required participants to be always sat on the saddle.
The V ̇O2max test comprised an initial 3-minute warm up period with no resistance (0 W) at a pace between 60 and 70 revolutions per minute (r•min −1 ).Each participant then started at 120 W, with 25 W increments every minute, participants were asked to cycle at exactly 80r•min −1 throughout the test.The test was terminated when cadence dropped by more than 10r•min −1 for more than 10-seconds despite strong verbal encouragement.Respiratory variables (RER, V ̇O2 , and CO 2 ) were measured continuously and averaged to 15-second intervals (Vyntus CPX; Vyaire Medical GmbH, Germany).Heart rate (HR) was recorded (Polar Electro Oy, Kempele, Finland) at every minute until volitional fatigue.The highest V ̇O2 value that was recorded was determined as V ̇O2max , whilst maximal external power output (W) was rounded down to the nearest incremental stage (Mean ± SD: 384 ± 60 W).Each participant's 55%Wmax relative intensity for the 1-hour submaximal endurance test were then calculated from these readings.

Familiarization (submaximal endurance test and 16.1 km TT).
Prior to commencement, participants' 55% relative intensity was loaded onto the monark 894e Peak Bike (mean ± sD: 209 ± 33 W), they were then instructed to cycle at 80r•min −1 for 1-hour.Respiratory variables were recorded continuously, averaged over 15-seconds and then averaged over 20-minute intervals for data analysis.heart rate and 20 µl capillary blood samples (glucose and lactate) were recorded and analyzed every 20-minutes (0, 20, 40, 60mins) (Biosen c-line sport, eKF diagnostic sales Gmbh, ebendorfer chaussee 3, Germany).Participants were then given a 5-minute rest period whereby water was consumed ad libitum (water consumption was recorded and standardized for the following visits) before starting the 16.1 km tt.During the tt, verbal encouragement was provided, and participants were blinded to all variables apart from distance elapsed.cycling performance was executed at a freely chosen cadence in order to complete the tt in the fastest time (seconds) possible.Oxygen consumption (ml•kg −1 •min −1 ) and power output (Watts) were measured throughout and consequently averaged for total mean comparative data analysis.capillary blood samples were also taken at every 4 km interval (4, 8, 12, 16 km).

Visit 2 Familiarization (lactate threshold test & repeated sprint performance test).
An incremental test performed at a constant 80r .min −1 starting at 120 W, with 25 W increments every 3-minutes was utilized.Respiratory variables were recorded continuously and averaged over 15-seconds.Capillary blood samples and HR were collected at rest and in the final 30-seconds of each 3-minute stage.The test was terminated once the participant could no longer maintain a cadence of 80r .min −1 for more than 10-seconds.Power output and HR during the first 4-stages and at the calculated LT were compared across each condition.Lactate threshold was calculated by using the Dmax method in 'Lactate E' software (Newell et al. 2007).
After a 10-minute rest, each participant was instructed to complete 3 × 20 second all out sprints on the Peak Bike.The RSPT was designed in accordance with previous work employing the same protocol (Gurney et al. 2021).Briefly, a 5-minute warm up at 50 W (including 2 × 3 second warm up sprints) was followed by a 1-minute recovery before starting 3 × 20 second all out sprints.The RSPT was performed with a flying start against an applied fixed load of 8.5% body mass which automatically engaged once the participant cycled above 90r .min −1 .Participants were blinded to all variables during the test.In between each sprint, participants were given a 4-minute active recovery period at 50 W where water was consumed ad libitum.Capillary blood samples were taken (lactate and glucose) in the 2 nd minute of each 4-minute recovery period.Average and peak power (W/kg), cadence (r .min −1 ), as well as fatigue indexes (Watts/sec & power drop %), were automatically recorded by the Peak Bike Monark software during each repeated sprint for analysis.
The first two visits were separated by 24 hrs, whereby participants completed the protocols in the exact same sequence as they would post supplement intervention.Participants were then randomly allocated to chlorella (Indigo Herbs Limitednutritional composition can be seen in Table 1) or placebo (microcrystalline cellulose) and were instructed to ingest 6-grams (14 capsules: five with breakfast, five with lunch, four with dinner) each day for 21-days.This daily dosage is similar to previous work (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017;Chidley and Davison 2018;Okada et al. 2018).The visually identical capsules were sealed within 21-small paper day bags and were coded by an independent lab technician.The morning after the 21-day supplementation, participants reported to the laboratory to begin their 2-day testing period (Visits 3 & 4).A minimum interval of 35-days (14-day wash out and the subsequent 21-day supplementation period) between the 4 th and 5 th visit was required before completing the exact same 2-day testing period (Visits 5 & 6).Participants failed to guess correctly which supplement they were on after the intervention, nor did they report any adverse gastrointestinal issues or taste differences.A schematic illustration of the protocol design can be seen in Figure 1.

Statistics
Data are presented as mean ± SD.All statistical procedures were carried out using IBM SPSS version 26 for windows.All data sets were analyzed for normality using Shapiro Wilk, while Mauchly's test of Sphericity was employed to establish any potential violations.Any violation of sphericity was corrected by using values from the Greenhouse-Geisser.Statistical significance alpha level was set at ≤.05.Effect size (ES) calculated using Partial ETA squared SPSS output, Observed Power and 95% Confidence Intervals (95% CI) were used where appropriate.All variables from the 1-hour submaximal endurance test variables, RSPT, HR and lactate during the first 4 stages of the LT tests (using the Dmax method) and the lactate values from the 16.1 km TT were analyzed using a two-way within subjects repeated measures ANOVA with a Bonferroni correction for multiple comparisons to determine any differences.Hemoglobin, LT and 16.1 km TT variables were compared using a paired sample T-Test.Individual responses for the 16.1 km TT and RSPT were also analyzed and compared using the smallest worthwhile change, (0.6 × 3.9% (CV 16.1 km TT)) and (0.5 × 2.5% (CV RSPT)) (Paton and Hopkins 2006).
Individual percentage change and performance times can be seen in Figure 4.There was no effect of condition or time for: lactate (p = 0.840), glucose (p = 0.256), HR (p = 0.663), power output (p = 0.594) and oxygen consumption (p = 0.556) during the TT (Table 2), and no condition*time interaction (p > 0.05).

Discussion
This is the first study to compare key physiological responses in trained cyclists during a submaximal endurance test, 16.1 km time trial, LT and RSPT after a 6 g/day  supplementation of CH for 21-days.The novelty of our results lies in the key findings; chiefly, chlorella supplementation resulted in a markedly improved relative power output during RSPTs, and significantly decreased lactate response during submaximal exercise intensities (Figure 2) and HR (Figure 3A).Collectively, these findings are similar to that of a previous study that investigated another popular microalgae (spirulina) with similar nutritional qualities (Gurney et al. 2021).Despite this, there are some differences in which constituents found in chlorella might also be contributing to any ergogenic aid benefit, which we discuss in further detail below.
Both average power and peak power (Figure 5) after the supplementation of chlorella yielded an average 5.4% improvement.Previous investigations have reported chlorella to be efficacious at improving maximal exercise bouts such as V ̇O2max in humans (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017) and maximal swimming bouts in rodents (Mizoguchi et al. 2011;Horii et al. 2017).Indeed, cyclists depend on the ability to produce short high intensity repetitive sprints whereby having a high relative power output is essential for a successful performance (Kordi et al. 2020;2020).Considering previous research has reported that neuromuscular and morphological changes are associated with improvements in PP performance (Dorel et al. 2005), and that our data demonstrated that cadence at the calculated PP was not significantly different between conditions, indicates that torque increased (Kordi et al. 2020) after chlorella supplementation.Mechanistically, the authors have limited comprehension to the exact constituents found in chlorella causing these findings due to the current novelty of chlorella supplementation research, and this despite previous authors also reporting that chlorella supplementation improves high intensity exercise (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017;Sanayei et al. 2021).Repeated sprinting with long recovery phases, such as the 4-minutes used in the present study, typically increases aerobic metabolism contribution (as much as 40%) during final sprint bouts with some athletes even reaching their  VO max 2 (Girard et al. 2011).Consequently, researchers have suggested that during the latter sprints, a greater aerobic contribution (by improving ones  VO max

2
) may minimize fatigue.Given that chlorella supplementation has previously improved aerobic capacity (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017;Sanayei et al. 2021) this could partly explain the increases in both PP and AP during the final sprint bout (Figure 5).These results could be owed to improvements in nutritional status (Umemoto and Otsuki 2014;Gurney and Spendiff 2022), or chlorella's anti-oxidant/inflammatory properties (Samadi et al. 2020).Despite little mechanistic evidence to support such findings, this applied performance data provides further evidence in the possible efficacy of chlorella supplementation and a foot hold for other researchers to explore and confirm the mechanisms.
Chlorella supplementation also reduced cardiac demand and lactate during submaximal cycling (Figures 2 and 3A/B).Chlorella has previously been reported to reduce arterial stiffness in young males, with the authors initially postulating their vasodilatory mechanistic findings to possible increases in NO production and bioavailability, believed to be derived from the arginine content in chlorella (Otsuki et al. 2013;Umemoto and Otsuki 2014).This speculation in the increase of NO production following 6 g/day chlorella supplementation was consequently confirmed by Otsuki et al. (Otsuki et al. 2015) who demonstrated that changes in arterial stiffness were correlated with increases in plasma nitrate/nitrite after four weeks.The ergogenic benefits of elevated nitrate/ nitrite on exercise performance are reviewed elsewhere (Pawlak-Chaouch et al. 2016), but briefly vasodilation derived from elevations in NO throughout the vasculature can facilitate an increased blood flow which therefore provides the working skeletal muscle with an increased peripheral oxygen offload and aid in the delivery/removal of key metabolites (Seeley et al. 2020), possibly allowing submaximal exercise bouts to be performed with a lower homeostatic disturbance (Figure 3A/B).Although arterial stiffness, blood pressure, and nitrate/nitrites mechanistic data collection were omitted due to being beyond the resources available for the current study, the homeostatic results from the submaximal cycle, coupled with the final repeated sprint bout, are further supportive that chlorella supplementation can improve aerobic capacity (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017).Importantly though, previous research on whether arginine itself may augment NO production (Álvares et al. 2012) and therefore positively affect aerobic exercise in healthy subjects is conflicting (Meirelles et al. 2019).Particularly as in the current study and previous chlorella supplementation studies (Otsuki et al. 2013;Umemoto and Otsuki 2014;Otsuki et al. 2015;Zempo-Miyaki et al. 2017) the daily intake of arginine was only 198 mg and 189 mg, respectively.Rather, it could be the combined consumption of bioactive properties found in chlorella such as PUFAs, glycoproteins, linoleic acid, antioxidants, and water-soluble fibers (Otsuki et al. 2013;Fallah et al. 2018) contributing to such improvements.It is therefore apparent that there are some conflicting mechanistic suggestions yet conversely there appears to be some uniformity in chlorella increasing nitrite/nitrate (Otsuki et al. 2015;Fujie et al. 2021).
To the best of our knowledge, this is the first study to investigate the influences of chlorella supplementation on lactate levels during exercise in humans.Recent reviews have reported that lactate is indeed a major fuel for mitochondrial respiration (Glancy et al. 2021).Lactate clearance capacity is often utilized as the gold standard for monitoring athlete training status (San-Millán et al. 2020).As such, if a lower lactate response is observed at the same given work rate (as observed in the current study -Figure 2).This may suggest an enhanced lactate metabolism and aerobic capacity.This is consistent with previous research in rodents where lower lactate concentrations were reported in the liver and muscle following chlorella supplementation (Mizoguchi et al. 2011;Horii et al. 2017).Horii et al. (Horii et al. 2017) demonstrated an enhanced MCT expression, lactate dehydrogenase activity, and proliferator-activated receptor ϒ coactivator-1α (PGC-1α) in rodents, key enzymes associated with lactate transport/ metabolism, energy metabolism, and mitochondrial biogenesis (Brooks 2018;2020).Consequently, these key adaptations will permit a lower lactate accumulation during exercise (Glancy et al. 2021), as observed by Horii and colleagues.Despite measurement and confirmation of increases in PGC-1α and MCT being beyond the scope of this study, increases in PGC-1α following chlorella supplementation have been translated and supported in a recent human study.Sanayei et al. (Sanayei et al. 2021) reported marginal increases in PGC-1α after supplementing obese women with chlorella (2.7 g/ day) combined with high-intensity interval training (3-sessions per week) for 8-weeks.Niacin, arginine, antioxidant properties of carotenoids, polyphenolic properties were the purported mechanistic constituents derived from chlorella contributed to these positive findings.It is evident that multiple constituents of chlorella may be acting concomitantly after supplementation, making true conclusive interpretation limited.
No differences were observed during the 16.1 km TT.Despite a 1.2% improvement in time, this variation typically falls within the expected race-race variation in road TTs (Paton and Hopkins 2006).Indeed, analysis of individual percentage change (Figure 4) revealed that only five participants improved their performance time above the calculated smallest worthwhile change.Further, the 16.1 km TT was performed at a substantially higher intensity and time above the calculated LT (Table 1 & LT results), considering results from previous research and the current study, the ergogenic effects of chlorella may only be seen during shorter, high intensity and intermittent exercise modalities (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017) and during intensities closer to the threshold which may explain the lack of significance.For example, during the first 4-lower-stages of the LT test, chlorella supplementation resulted in no differences in the blood lactate response.This data also supports our findings from the 1-hour submaximal test (207 W, −81 W below the calculated average lactate threshold when using the Dmax method) where during the first 40-minutes of steady state submaximal cycling there were no differences in lactate.Equally, at intensities much higher than the threshold (RSPT) chlorella also had no effect on lactate.However, during the latter end of the 1-hour submaximal test, where HR values were similar to the calculated threshold (144 ± 12b .min −1 ) a significant difference in lactate occurred.These findings are perhaps indicative that there may be a specific intensity whereby chlorella supplementation in trained cyclists may exhibit improvements in metabolic perturbation.The non-significant results during the 16.1 km TT are also similar to the previous paper in spirulina (Gurney et al. 2021).Currently the literature on chlorella's influence on lactate metabolism in humans is sparse, thus making conclusive comparisons from previous research difficult.However, Horii et al. (Horii et al. 2017) demonstrated that chlorella supplementation in rats is most effective at influencing lactate metabolism whilst employing a high intensity training programme rather than supplementation alone.To elucidate why this might be, future research should consider the possibility of supplementing chlorella alongside a controlled training programme whilst comparing LTs in humans.It's also important to consider a key limitation to our LT test as it was completed on the second day of testing, 24 hrs after fatiguing exercise, therefore our findings might not necessarily be translatable or ecologically valid due to the majority of cyclists usually completing a LT in a fresh state.
Based on the literature to date, this may be the first study to demonstrate a small positive hemopoietic trend following chlorella supplementation in trained males.It could be argued that the negligible, yet significant, 4% improvement in Hb following chlorella supplementation in comparison to placebo may have also had a small influence on exercise performance parameters in this study.The CV% for this variable was 1.2% and the smallest worthwhile change in Hb was calculated at being 2.06 g/L (0.2 X STD of chlorella (10.3 g/L) (Hopkins 2000).Overall, there was a 6 g/L increase from placebo to chlorella, which according to previous literature may equate to a 2% improvement in oxygen-carrying capacity in the blood (Otto et al. 2013).Indeed, although not significant, there was a 2.5% reduction in the demand for oxygen following chlorella supplementation (34.0 ± 3.9 ml•kg −1 •min −1 ) in comparison to placebo (34.9 ± 5.5 ml• kg −1 •min −1 ) during the submaximal cycling test.These findings in turn, may support how chlorella supplementation has previously improved maximal oxygen uptake (Umemoto and Otsuki 2014;Zempo-Miyaki et al. 2017) and exhibited lower homeostatic disturbances during submaximal cycling in the current study (lower lactate (Figure 2) and HR (Figure 3).It's plausible to suggest that the iron content, combined with the folate found in chlorella contributed to this small change in Hb after supplementation (Nakano et al. 2010).
This study acknowledges several key limitations and therefore interpretation of the results should come with caution.Firstly, the primary aim of this study was to solely focus on cycling performance parameters following chlorella supplementation, mechanistic results to support such findings are therefore missing.Further mechanistic research is warranted to explore the speculations, as currently conclusive interpretation of the results is limited.Secondly, although we controlled for food intake prior to testing day 1, we did not account for individual food and recovery strategies the 24 hrs in between testing day 1 and 2. Additionally, we asked participants to consume the capsules with each meal per day throughout the intervention, as we did not assess individual food/eating habits, there may have been disparities in the timing/omission of meal consumption throughout the day.There may also be possible interactions with certain food stuffs which may influence the bioavailability/absorption of the micronutrients in microalgae (Koyande et al. 2019), understanding of which is currently unknown.Thirdly, the 16.1 km TT may limit the applicability of the results as it may be considered a relatively short 'sprint-like' distance for experienced cyclists, it is unknown as to whether a longer endurance distance (such as 50+ km) would derive the same results.Finally, while participants in the current study were considered fit healthy men who train regularly, prior and post analysis of their nutritional status was not conducted.We also acknowledge that third-party independent nutrient analysis was not conducted on the chlorella supplement.
Collectively, the novel findings of this study indicate that 6 g/day chlorella supplementation for 21-days can significantly improve relative power during RSPTs (Figure 5) and allow submaximal exercise to be performed with lower homeostatic disturbances.Conversely, chlorella does not improve 16.1 km TT and LT's.Chlorella may therefore pose as an additional supplement for cyclists to consider, particularly for those cyclists who want to improve their sprinting.

Figure 1 .
Figure 1. a Schematic illustration of the protocol design.

Figure 4 .
Figure 4. participant percentage change in performance (%) between placebo and chlorella during the 16.1 km tt (a). the dotted grey lines indicate the smallest worthwhile change in performance.average and individual performance time (secs) (b).

Figure 5 .
Figure 5. average power (a) and peak power (b) (watts/kg), mean ± SD of each repeated sprint performance test following the 21-day supplementation of chlorella or placebo.* signifies p < 0.05.†# signifies both a chlorella ( †) and placebo (#) within trial increase p < 0.05.individual average (c) and peak power performance percentage change (d) between placebo and chlorella.the dotted grey lines indicate the smallest worthwhile change in performance.the solid black line indicates the group mean.

Table 2 .
mean ± SD for performance variables measured across the 16.1 km tt.