Effects of d-allulose intake during exercise training on heart rate response to submaximal exercise in untrained young adults: a pilot study

Abstract

[Purpose]

d-Allulose has been reported to enhance fat oxidation during acute aerobic exercise; however, its effects during exercise training in humans remain unclear. We investigated the effects of d-allulose intake during exercise training on physiological responses to submaximal exercise in untrained young adults.

[Methods]

Eighteen untrained young adults were randomly assigned to receive a beverage with (ALLU) or without (PLA) 5 g of d-allulose 30 min before exercise. Participants exercised on a cycle ergometer at 50% of their estimated $\dot{V}$O_2max for 30 min, 4 days per week, for 4 weeks. Heart rate at the end of each session was recorded as an indicator of physiological responses to submaximal exercise. Body composition and estimated $\dot{V}$O_2max were measured before and after the intervention.

[Results]

No significant between-group differences were observed in body composition. The relative change in estimated $\dot{V}$O_2max tended to be greater in the ALLU group than in the PLA group, although this difference was not statistically significant (p = 0.09). The temporal pattern of absolute heart rate differed significantly between groups over the 4-week intervention period (treatment × week interaction, p = 0.007), with no significant between-group differences observed at individual weeks. Exploratory analysis showed that relative heart rate was significantly lower at Week 4 in the ALLU group than in the PLA group (p = 0.028).

[Conclusion]

Repeated d-allulose supplementation before exercise during 4 weeks of training was associated with a different temporal pattern of heart rate responses to submaximal exercise in untrained young adults. These preliminary findings warrant further investigation into the effects of d-allulose supplementation during exercise training.

INTRODUCTION

Cardiorespiratory fitness, indicated by maximal oxygen uptake ( $\dot{V}$O_2max), is associated with elite performance in endurance sports [1] and all-cause mortality in the general population [2]. Substantial evidence indicates that a higher $\dot{V}$O_2max is associated with a lower risk of several lifestyle-related diseases [3]. Although $\dot{V}$O_2max is considered a key indicator of cardiorespiratory fitness, its direct measurement requires specialized equipment for respiratory gas analysis and is not always feasible in practical exercise settings. In contrast, heart rate can be measured easily and repeatedly during submaximal exercise, making it a practical indicator for evaluating physiological responses to a standardized workload. Endurance training has been shown to reduce heart rate during exercise at a constant submaximal workload, reflecting physiological adaptation to repeated exercise training [4-6]. Therefore, heart rate during submaximal exercise may provide a practical index of cardiovascular adaptation to exercise training, although heart rate alone cannot identify the underlying physiological mechanisms.

Nutritional strategies, such as ergogenic aids, are commonly used to improve athletic performance and health-related physical fitness [7]. Several nutritional supplements have been reported to improve endurance capacity [8]. Functional foods that can enhance physical fitness and promote health, have gained popularity. Since the effects of exercise and nutritional supplementation are often additive, verifying their combined effects is important for effectively improving physical fitness, particularly endurance.

d-Allulose (or d-psicose) is a rare sugar present in limited quantities in nature. d-Allulose is a C-3 epimer of d-fructose with approximately 70% of the sweetness of sucrose and almost zero calories. In both rodents and humans, d-allulose attenuates postprandial blood glucose levels [9-12], reduces abdominal fat accumulation [13-15], and alleviates insulin resistance and improves glucose tolerance [16-18] through multiple mechanisms. d-Allulose can inhibit α-glucosidase activity [12], stimulate hepatic glycogen synthesis via glucokinase activation [11,19], and promote the secretion of glucagon-like peptide-1 from intestinal L-cells to regulate glucose concentrations after carbohydrate intake20. d-Allulose also modulates lipid metabolism by suppressing the activation of genes involved in fatty acid synthesis, thereby enhancing lipolysis [21,22].

Recently, we reported that d-allulose administration enhanced endurance and reduced fatigue in mice [23]. Additionally, findings from our previous study indicated that combining exercise with d-allulose intake effectively upregulates endurance capacity in mice [24]. In a study involving human participants, d-allulose intake prior to aerobic exercise was reported to increase fat oxidation during exercise [25]. However, most human studies have examined the acute effects of d-allulose, whereas the effects of repeated d-allulose supplementation during an exercise training program on physiological responses to exercise remain largely unknown. Therefore, clarifying whether the combination of exercise training and d-allulose supplementation can be an effective intervention for improving physical responses may contribute to the development of strategies for enhancing endurance performance and promoting health. Hence, this study aimed to investigate the effects of d-allulose intake combined with exercise training on physical responses to submaximal exercise in untrained young adults.

METHODS

Participants

All experimental procedures were approved by the Ethics Committee of Meijo University (R5-2) and were conducted in accordance with the Declaration of Helsinki. Untrained, healthy young college students were recruited by word of mouth and informed of the potential risks associated with the intervention and measurements. All participants provided written informed consent to participate in this study. Participants were excluded if they had engaged in regular aerobic or resistance training for at least 1 year prior to the start of the study, were taking any dietary supplements, functional foods, or medications, or had chronic diseases as assessed by medical history. Participants were randomly assigned to either the placebo (PLA) or d-allulose (ALLU) group using a computer-generated randomization sequence created with the RAND function in Microsoft Excel by a research collaborator who was not involved in participant recruitment or outcome assessment. Randomization was stratified by sex, with separate computer-generated allocation sequences prepared for male and female participants. The allocation sequence was retained by the research collaborator and was inaccessible to the investigator responsible for participant enrollment. Both the participants and the investigators responsible for outcome assessments remained blinded to group assignments until completion of the study. Baseline characteristics of the participants are shown in Table 1. Participants were instructed to maintain their usual dietary and physical activity habits, avoid binge eating, and refrain from taking any dietary supplements or functional foods during the experimental period. Although dietary and physical activity surveys were not conducted in this study, participants did not report major changes in their lifestyle during the experimental period.

Table 1.

Baseline characteristics of the participants

	PLA	ALLU	p-value
Age (years)	18.8 ± 0.7	18.7 ± 0.5	0.694
Sex (M / F)	3 / 6	4 / 5	1.000
Height (cm)	161.4 ± 7.2	161.7 ± 8.6	0.935
Body weight (kg)	53.4 ± 8.7	52.0 ± 4.7	0.674
BMI (kg/m2)	20.4 ± 2.0	20.0 ± 2.0	0.657
% body fat (%)	21.8 ± 7.7	19.8 ± 9.0	0.609
LBM (kg)	42.0 ± 9.3	41.8 ± 6.1	0.955
Muscle mass (kg)	39.7 ± 8.9	39.5 ± 5.9	0.951

Values are expressed as means ± SD. PLA, placebo; ALLU, d-allulose; BMI, body mass index; LBM, lean body mass. The p-values were obtained using unpaired Student's t-tests for continuous variables and Fisher’s exact test for sex distribution.

Overview of Experimental Design

This study was designed as a pilot randomized, double-blind, placebo-controlled trial. Although the study was approved by the Ethics Committee of Meijo University prior to participant recruitment, it was not prospectively registered in a public clinical trial registry. Before the trial, body weight, body fat percentage, lean body mass (LBM), and muscle mass were measured via bioelectrical impedance analysis using a body composition analyzer (MC-980A, Tanita, Tokyo, Japan). Body height was measured using a height meter only during the pre-assessment. Additionally, $\dot{V}$O_2max was estimated using a programmed incremental-load exercise test on a cycle ergometer (Aerobike 75XLⅢ, Konami Sports Life Co., Ltd., Tokyo, Japan). The pre- and post-measurements were performed in the afternoon, at least 2 h after a meal, and each exercise test was scheduled at the same time of day for each participant. Participants exercised on a cycle ergometer at 50% of their estimated $\dot{V}$O_2max for 30 min, 4 days per week, for 4 weeks. Participants ingested a commercially available beverage (250 mL/bottle) with (ALLU) or without (PLA) 5 g of d-allulose 30 min before each daily exercise bout. They consumed the beverages within 1 min under the supervision of the researchers. After the 4-week intervention, body composition and estimated $\dot{V}$O_2max were measured again. The primary outcome was absolute heart rate during submaximal exercise. Secondary outcomes included estimated $\dot{V}$O_2max, body composition, blood glucose and lactate levels.

Test and Placebo Beverage

Commercially available sports drinks (250 mL/bottle) and d-allulose were provided by Matsutani Chemical Industry Co., Ltd. (Hyogo, Japan). d-Allulose (5 g) was dissolved in the test beverage. A previous human study used the same dose of d-allulose and reported that its administration 30 min before exercise increased fat oxidation during exercise25. Commercially available sports drinks were used to mask the sweet taste of d-allulose, and the beverages could not be distinguished based on appearance, sweetness, or flavor. To equalize the calorie content, no additional ingredients were added to the placebo beverage because d-allulose contains zero calories. These beverages were prepared by a research assistant so that the participants and the principal researcher were blinded to the beverage composition. Sequence allocation concealment and the blinding of the participants and the principal researcher were maintained until all post-intervention measurements were completed. The nutrient components per serving (250 mL) of both beverages are listed in Table 2.

Table 2.

Nutrient components of the experimental beverages

Contents	PLA	ALLU
Energy (kcal)	62.5	62.5
Carbohydrate (g)	15.5	15.5
Protein (g)	0	0
Fat (g)	0	0
Sodium (g)	0.3	0.3
Potassium (mg)	50	50
Calcium (mg)	5	5
Magnesium (mg)	1.5	1.5
d-Allulose (g)	0	5

PLA, placebo; ALLU, d-allulose.

Incremental Load Exercise Test and Exercise Training

The participants performed an incremental-load exercise test on a cycle ergometer (Aerobike 75XLⅢ) using a programmed physical fitness test mode to estimate $\dot{V}$O_2max before and after the intervention. The exercise test consisted of 1 min of rest followed by approximately 10 min of an incremental ramp exercise test at 50 rpm. Heart rate (bpm) was measured using an ear sensor attached to the ergometer, and the test ended when the heart rate reached 75% of the age-predicted maximum heart rate (HR_max). HR_max was predicted using the equations 209 − 0.69 × age (years) in men and 205 − 0.75 × age (years) in women. $\dot{V}$_O2 was calculated using a regression equation between workload and heart rate obtained during each participant’s measurement and a workload- $\dot{V}$_O2 equation programmed into the cycle ergometer. The workload at the age-predicted HR_max was calculated as the maximum workload (W_max), and the $\dot{V}$_O2 at W_max was calculated as the estimated $\dot{V}$O_2max using the regression equation (estimated $\dot{V}$O_2max = 9.386 × W_max + 289.6), as previously described using the same cycle ergometer model [26,27]. The exercise workload was individually determined based on 50% of the estimated $\dot{V}$O_2max measured before the intervention and was maintained unchanged throughout the 4-week intervention (PLA: 73 ± 26 W, ALLU: 74 ± 22 W).

The exercise intensity was set at 50% of estimated $\dot{V}$O_2max because moderate-intensity exercise (approximately 40-50% $\dot{V}$O_2max) has been shown to improve cardiorespiratory fitness in previously untrained individuals while providing a constant submaximal workload suitable for assessing changes in heart rate responses [28-30]. The participants performed exercise training on a cycle ergometer at the target exercise intensity for 30 min, 4 days per week, for 4 weeks. Heart rate was monitored during exercise using an ear sensor attached to the ergometer and was recorded at the end of each daily exercise bout. During the first and final bouts of daily exercise, a small amount of blood was collected from the fingertip, and blood glucose and lactate levels were measured using a Glutest Neo Super device (Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan) and a Lactate Pro 2 device (Arkray, Kyoto, Japan) before beverage consumption, immediately before exercise, 5 min after beginning exercise, and at the end of exercise.

Statistical Analysis

As this study was designed as a pilot study, no formal sample size calculation was performed. The sample size was determined based on feasibility and recommendations for pilot studies [31]. Statistical analyses were performed on a per-protocol basis. Data are presented as means ± standard deviation (SD). Baseline continuous variables, including age, height, and body composition variables, were compared between groups using unpaired Student’s t-tests, whereas sex distribution was compared using Fisher’s exact test. The relative value of estimated $\dot{V}$O_2max was calculated as [(post value / pre value) × 100] and compared between groups using an unpaired Student’s t-test. For each participant, absolute heart rate for each week was calculated as the mean of the heart rate values recorded at the end of the four exercise sessions performed during that week. Relative heart rate was calculated by normalizing each weekly absolute heart rate to the Week 1 value, with the Week 1 value set to 100%, and was analyzed as an exploratory outcome. Body composition and estimated $\dot{V}$O_2max were analyzed using two-way mixed-design ANOVA with treatment (PLA vs. ALLU) as the between-participant factor and session (Pre vs. Post) as the within-participant factor. Absolute heart rate across Weeks 1-4 was analyzed using a mixed-effects model with treatment and week as fixed effects and participant as a random effect. The mixed-effects model was fitted using restricted maximum likelihood. Relative heart rate was analyzed exploratorily using two-way mixed-design ANOVA with treatment as the between-participant factor and week as the within-participant factor. Bonferroni’s post hoc comparisons were performed where appropriate. Glucose and lactate levels were analyzed using three-way mixed-design ANOVA with treatment as the between-participant factor and session and time (pre-beverage, pre-exercise, 5 min after exercise onset, and end of exercise) as the within-participant factors. Normality of the residuals was assessed by visual inspection of Q-Q plots. For within-participant factors with more than two levels, the Geisser-Greenhouse correction was applied when sphericity could not be assumed. Sphericity was not applicable to within-participant factors with only two levels. Effect sizes for ANOVA and mixed-effects model fixed effects were expressed as partial eta squared (partial η²), calculated from the F statistic and the corresponding numerator and denominator degrees of freedom. For key between-group comparisons, mean differences and their 95% confidence intervals (CIs) were also reported. Statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism software (version 10.2.1; GraphPad, San Diego, CA, USA).

RESULTS

Study Participants

Twenty-two untrained, healthy college students (18.8 ± 0.6 years; 10 men and 12 women) volunteered for this pilot trial. One participant was excluded based on the exclusion criteria. The remaining 21 participants were randomly allocated to two groups. Three additional participants withdrew before completing the intervention because of acute infectious illnesses (COVID-19 or influenza) that were considered unrelated to the study, resulting in a final sample of 18 participants (Figure 1). All 18 participants completed all scheduled exercise sessions. Beverage compliance among these participants was 100%. Baseline characteristics of the three participants who withdrew were descriptively compared with those of the 18 completers, and no marked differences were observed.

Figure 1. Flow diagram of study participants.

ALLU, d-allulose; PLA, placebo.

Effects of d-Allulose intake with exercise training on body composition

Body composition before and after the intervention is shown in Table 3. Two-way mixed-design ANOVA (treatment × session) revealed no significant main effects or interactions for body weight, body mass index, body fat percentage, LBM, or muscle mass.

Table 3.

Changes in body composition of the participants

				p-value (partial η²)
		PLA	ALLU	Treatment	Session	Interaction
Body weight (kg)	Pre	53.4 ±8.7	52.0 ± 4.7	0.682 (0.011)	0.340 (0.057)	0.883 (0.001)
	Post	53.7 ± 8.5	52.4 ± 4.8
BMI (kg/m2)	Pre	20.4 ± 2.0	20.0 ± 2.0	0.646 (0.014)	0.672 (0.011)	0.982 0.000
	Post	20.5 ± 2.0	20.1 ± 1.9
% body fat (%)	Pre	21.8 ± 7.7	19.8 ± 9.0	0.673 (0.011)	0.220 (0.092)	0.399 (0.045)
	Post	22.0 ± 7.5	20.8 ± 8.4
LBM (kg)	Pre	42.0 ± 9.3	41.8 ± 6.1	0.925 (0.001)	0.874 (0.002)	0.761 (0.006)
	Post	42.0 ± 9.0	41.5 ± 6.2
Muscle mass (kg)	Pre	39.7 ± 8.9	39.5 ± 5.9	0.926 (0.001)	0.843 (0.003)	0.802 (0.004)
	Post	39.7 ± 8.5	39.3 ± 5.9

Values are expressed as means ± SD. PLA, placebo; ALLU, d-allulose; BMI, body mass index; LBM, lean body mass.

Effects of d-Allulose intake with exercise training on physical fitness

Two-way mixed-design ANOVA (treatment × session) revealed no significant interaction for mean estimated $\dot{V}$O_2max (F(1,16) = 2.856, p = 0.110, partial η² = 0.151) (Figure 2A). Although the relative value of estimated $\dot{V}$O_2max was slightly greater in the ALLU group than in the PLA group, this difference did not reach statistical significance (mean difference [ALLU minus PLA], 12.21 percentage points; 95% CI, −2.54 to 26.96; p = 0.09) (Figure 2B). A mixed-effects model of weekly mean absolute heart rate revealed a significant treatment × week interaction (F(3,48) = 4.576, p = 0.007, partial η² = 0.222) and a significant main effect of week (F(2.783,44.53) = 9.670, p < 0.001, partial η² = 0.377), whereas the main effect of treatment was not significant (F(1,16) = 0.421, p = 0.526, partial η² = 0.026) (Figure 3A). Post hoc comparisons showed no significant between-group differences in absolute heart rate at any individual week. In an exploratory analysis, relative heart rate was calculated by normalizing each participant’s weekly heart rate to the Week 1 value. A two-way mixed-design ANOVA of relative heart rate revealed a significant treatment × week interaction (F(3,48) = 4.776, p = 0.005, partial η² = 0.230) and significant main effects of week (F(2.861,45.77) = 8.988, p < 0.001, partial η² = 0.360) and treatment (F(1,16) = 5.143, p = 0.038, partial η² = 0.243). Post hoc analysis revealed that relative heart rate at Week 4 was significantly lower in the ALLU group than in the PLA group, with a between-group mean difference (ALLU minus PLA) of −6.04 percentage points (95% CI, −11.53 to −0.56; adjusted p = 0.028) (Figure 3B).

Figure 2. Effects of d-allulose intake with exercise training on physical fitness.

Estimated maximum oxygen uptake ( $\dot{V}$O_2max) (A) was indirectly measured as an index of physical fitness using a programmed incremental-load exercise test on a cycle ergometer before and after the 4-week intervention. The relative value of estimated $\dot{V}$O_2max (B) was calculated as [(post value / pre value) × 100]. Values are shown as means ± standard deviation.

Figure 3. Effects of d-allulose intake with exercise training on heart rate throughout the intervention.

Heart rate during daily exercise was monitored and recorded at the end of a daily exercise bout. Absolute heart rate values (A) represent the weekly mean of heart rate values recorded at the end of each of the four exercise sessions. In an exploratory analysis, relative heart rate values (B) were calculated by normalizing each weekly mean absolute heart rate to the Week 1 value, with the Week 1 value set to 100%. Values are presented as means ± standard deviation. * p < 0.05, PLA vs. ALLU.

Effects of d-Allulose intake on blood glucose and lactate levels during an exercise bout

To evaluate whether d-allulose intake affects blood energy substrates during exercise, we measured blood glucose and lactate levels during the initial and final exercise bouts. Three-way mixed-design ANOVA (time × session × treatment) indicated a significant main effect of time on glucose levels (F(3, 48) = 95.53, p < 0.001, partial η² = 0.857). Blood glucose levels increased after beverage consumption and decreased during exercise; however, no significant differences were observed between the PLA and ALLU groups before or after the intervention (Figure 4A). The three-way mixed-design ANOVA (time × session × treatment) also indicated a significant time × session interaction (F(2.085, 33.36) = 8.1, p = 0.001, partial η² = 0.336) and significant main effects of time (F(3.000, 48.000) = 12.03, p < 0.001, partial η² = 0.429) and session (F(1, 16) = 14.2, p = 0.002, partial η² = 0.470) on blood lactate levels. Blood lactate levels increased 5 min after exercise onset in both groups before the intervention; however, this increase was attenuated in both groups after the intervention, with no significant difference between the groups (Figure 4B).

Figure 4. Effects of d-allulose intake on blood glucose and lactate levels during an exercise bout.

Blood glucose (A) and lactate levels (B) were measured before drinking the beverage consumption (0 min), immediately before exercise (30 min), 5 min after beginning exercise (35 min), and at the end of exercise (60 min). Values are presented as means ± standard deviation.

DISCUSSION

The principal finding of the present pilot study was that repeated d-allulose supplementation was associated with a different temporal pattern of the heart rate response to submaximal exercise during the 4-week training intervention. In contrast, no statistically significant between-group difference was observed in estimated $\dot{V}$O_2max. To our knowledge, few human studies have investigated the potential effects of repeated d-allulose supplementation during exercise training on physiological responses to exercise.

d-Allulose has been accepted as a food with function claims in Japan because it has been reported to suppress postprandial blood glucose increases and enhance fat oxidation. Although d-allulose reduces body weight and fat mass in various animal models of obesity, similar effects have not been demonstrated in humans [32,33]. This study found no significant changes in average body composition. In contrast to the current study, a previous study reported weight loss following d-allulose intake using higher doses (twice-daily doses of 4 g or 7 g) and a longer intervention duration (12 weeks) [15].

The exercise intensity used in the present study was selected based on previous reports demonstrating that moderate-intensity exercise (approximately 40-50% $\dot{V}$O_2max) improves cardiorespiratory fitness in untrained individuals [28-30]. Nevertheless, no significant improvement in estimated $\dot{V}$O_2max was observed in the present study, although the ALLU group showed a slightly greater relative increase than the PLA group. This finding should be interpreted with caution because $\dot{V}$O_2max was estimated using a heart-rate-based algorithm incorporated into the cycle ergometer rather than being directly measured by respiratory gas analysis. Therefore, the observed slight difference in estimated $\dot{V}$O_2max should be considered exploratory and requires confirmation using direct measurements of oxygen uptake in future studies.

Regarding energy metabolism, increased fat oxidation and reduced carbohydrate utilization may contribute to muscle glycogen sparing and endurance capacity. In rodent studies, d-allulose feeding for 4 weeks increased energy expenditure and fatty acid oxidation [34]. Our previous research in trained mice also demonstrated that d-allulose intake suppressed muscle glycogen depletion during exercise and was associated with enhanced endurance capacity [24]. In humans, Kimura et al. [35] reported that d-allulose enhanced postprandial fat oxidation in a randomized, single-blind crossover trial. Yamaguchi et al. [25] also reported that d-allulose intake prior to aerobic exercise for 60 min increased fat oxidation during exercise; however, whether d-allulose intake combined with exercise training affects endurance-related physiological responses in humans remains unclear. Given these previous findings, one possible hypothesis is that alterations in substrate utilization, including enhanced fat oxidation and glycogen sparing, may have contributed to the different temporal pattern of heart rate responses observed in the present study. In an exploratory analysis, relative heart rate at Week 4 was also significantly lower in the ALLU group than in the PLA group. However, energy substrate utilization and cardiovascular responses were not directly assessed in the present study. Furthermore, blood glucose and lactate responses did not differ between the groups. Therefore, the involvement of altered substrate utilization in the observed heart rate response remains speculative and cannot be established from the present data.

d-Allulose can attenuate postprandial blood glucose levels in animals and humans; however, no difference was observed in changes in blood glucose levels following beverage intake and during exercise between the placebo and d-allulose conditions. Moreover, the increase in lactate concentration 5 min after exercise initiation was attenuated after the 4-week intervention, but no effect of d-allulose intake on lactate levels was observed. Since the participants were untrained, it is likely that the lower blood lactate levels observed after the intervention were likely attributable to improvements in the oxidative capacity of peripheral tissues (e.g., skeletal muscle) induced by exercise training rather than by d-allulose intake. In addition, blood glucose and lactate levels were assessed only during the first and final exercise bouts; therefore, temporal changes in metabolic responses throughout the 4-week intervention could not be evaluated. Thus, the observed glucose and lactate responses do not appear to explain the difference in heart rate between the groups.

This study has several limitations. First, the sample size was small, and although both male and female participants were included, the number of participants was insufficient to permit meaningful sex-stratified or sex-adjusted analyses. In addition, menstrual cycle phase was not recorded in female participants. Previous studies have demonstrated sex differences in cardiovascular and substrate metabolic responses to exercise, and menstrual cycle-related fluctuations in ovarian hormones may also influence physiological responses to exercise, including heart rate responses [36-38]. Therefore, the potential influence of sex and menstrual cycle phase on the observed effects of repeated d-allulose supplementation cannot be excluded. Future randomized controlled trials with larger and more balanced samples should incorporate prospective assessment of menstrual cycle phase and recruit sufficient numbers of male and female participants to clarify potential sex-specific responses to d-allulose supplementation. Second, $\dot{V}$O_2max was not directly measured but estimated from heart rate, and thus the results related to aerobic capacity should be interpreted with caution. Third, heart rate was measured using an ear-mounted sensor, which may be less accurate than electrocardiography or chest strap methods during exercise. However, heart rate was measured under standardized conditions using the same method throughout the intervention for each participant. Fourth, d-allulose or placebo was ingested 30 min before every exercise session, including the sessions in which heart rate was assessed. Therefore, the present study design cannot distinguish whether the observed difference in the temporal pattern of heart rate reflects a chronic effect of repeated d-allulose intake during exercise training, an acute effect of d-allulose ingestion before exercise, or a combination of both. Future studies should include post-training exercise assessments without acute d-allulose ingestion to distinguish chronic training-related adaptations from acute effects. Fifth, although participants were instructed to maintain their habitual dietary intake and physical activity throughout the intervention, neither dietary intake nor physical activity was objectively monitored. Therefore, the potential influence of uncontrolled lifestyle factors on the observed outcomes cannot be excluded. Future studies should include dietary records and objective assessments of habitual physical activity. Sixth, although absolute heart rate during submaximal exercise was identified as the primary outcome, multiple outcomes were analyzed without adjustment for multiplicity across outcomes. In particular, the analysis of relative heart rate, including the Week 4 between-group comparison, was exploratory and should be interpreted cautiously given the small sample size and the lack of prospective specification. Because relative heart rate was normalized to the Week 1 value, the potential influence of the numerical between-group difference in absolute heart rate at Week 1 and regression to the mean cannot be excluded. In addition, although the mixed-effects model of absolute heart rate showed a significant treatment × week interaction, the model did not explicitly adjust for the numerical between-group difference in heart rate at Week 1. Therefore, the observed interaction should be interpreted cautiously as reflecting a difference in the temporal pattern of heart rate responses between the groups. Finally, the mechanisms underlying the observed difference in heart rate responses remain unclear because energy substrate utilization and detailed cardiovascular responses during exercise were not directly assessed. Therefore, further randomized controlled trials with larger sample sizes and more precise physiological measurements are required to confirm and extend these findings.

From a practical perspective, repeated d-allulose supplementation may warrant further investigation as a potential adjunct to exercise training, particularly for sedentary individuals or those beginning an exercise program. The different temporal pattern of heart rate observed between the groups provides a rationale for further investigation of d-allulose supplementation in the context of exercise training. However, because the present study was a pilot trial involving healthy young adults, these potential implications remain speculative and should be evaluated in larger studies including diverse populations.

In conclusion, repeated d-allulose supplementation before exercise during a 4-week training intervention was associated with a different temporal pattern of heart rate response to submaximal exercise in untrained young adults. Given the pilot nature and small sample size of this study, further adequately powered randomized controlled studies incorporating direct physiological measurements are warranted to confirm these findings and better characterize the effects of d-allulose during exercise training.