**1. Introduction**

The importance of pre-exercise nutrition for exercise performance has been well documented [1–6]. However, given that many competitive endurance activities (training and/or competition) are scheduled early in the morning, there exists a major limitation: inadequate time in the morning prior to the event to properly fuel for sport. In addition, endurance athletes seldom consume much, if anything, before training or competitions of 75–90 min in duration [7–11]. Unfortunately, this behavior may result in sub-optimal physiological conditions such as carbohydrate depletion, dehydration, and fatigue [12], which will adversely impact training quality and performance. This issue highlights the need to

develop strategies to provide adequate nutrition from foods, beverages, and/or supplements that athletes can consume pre-sleep without inducing gastrointestinal distress (GID) or disrupting normal sleep patterns [6,8]. As an intervention, the incorporation of a pre-sleep meal may provide an added "window of opportunity" for optimizing next-morning pre-race carbohydrate (CHO) availability and exercise performance.

Glucose is the body's preferred energy substrate during endurance exercise. Currently, high glycemic index (HGI) CHOs are utilized by most athletes for pre- and/or intra-exercise nutrition due to their rapid breakdown which drastically increases blood glucose availability. However, complications such as GID, may arise with the consumption of HGI CHO sources prior to or during exercise due to the gastrointestinal sensitivity to nutrient intake [13]. Consequently, this raises the question as to the role of low glycemic index (LGI) CHO for exercise performance and nutrient timing. Contrary to a HGI, a LGI slowly digests carbohydrates, thus providing more stable and long-lasting glucose release, which may lower GID, both of which may better support endurance exercise performance [14].

Previous literature agrees that consuming LGI CHO prior to exercise results in enhanced fat oxidation [15–18], and likely improved exercise performance [14,19]. However, not all studies agree [20–23]. Stevenson and colleagues [22,23] investigated the effects of a low vs. high glycemic index evening meal, approximately 16 h prior (at 19:00 p.m.), on next-morning metabolic responses at rest and during exercise in males [23] and females [22]. Though in these studies [22,23] participants were fed a standard HGI breakfast (at 08:00 a.m.) three hours prior to a 60-min run at 65% VO2max. Although the breakfast elicited immediate post-prandial effects (lower glycemic and insulinemic responses), there was no significant effect of the previous evening's dinner glycemic index on substrate utilization at rest or during running [22,23]. Though, given that meals were consumed 16 h prior to testing, and participants were given a standardized HGI breakfast in the morning, it is not surprising that any residual metabolic effects could not be detected. Given these limitations, it remains unknown if pre-sleep CHO supplementation can optimize next-morning endurance athlete fuel selection and performance, and whether low or high glycemic index would be preferential.

Though a LGI is touted as an efficacious source of CHO, a novel hydrothermally modified LGI starch supplement was developed to manage glucose levels by providing a slow and steady release of glucose to the body and brain for up to ten hours at a time [24]. In fact, data indicate a lower peak and less rapid rate of decline in blood glucose than conventional cornstarch, which is already considered a LGI CHO [25,26]. Currently, only a few studies have included the use of this novel LGI CHO supplement [15,17,21,27] all of which included supplement ingestion either before, during, or after exercise. No studies have investigated the effects of pre-sleep ingestion of a modified CHO on next-morning exercise metabolism and performance. However, there is a plethora of data investigating protein pre-sleep. The only study, to date, that investigated pre-sleep CHO-type beverage was conducted by Ormsbee et al. (2016) [28]. The authors investigated the effect of pre-sleep chocolate milk (HGI and protein) on endurance performance and found that chocolate milk resulted in increased carbohydrate oxidation in the morning, but effects did not translate to 10-km running performance improvements in females [28]. Given these data, we want to explore the effects of pre-sleep LGI CHO since it has the potential to have a positive impact based on the slow/long-lasting release of glucose, the lasting satiety, and if a HGI increases CHO oxidation, it is reasonable to suspect that LGI CHO would have the opposite effect, as noted in acute day of studies.

Accordingly, the purpose of this study was to determine the effects of nighttime pre-sleep supplementation with a novel LGI CHO on the next morning: (1) resting metabolism and GID; (2) metabolic and GID responses to incremental exercise; and (3) 5-km time trial running performance in trained male and female endurance athletes. It was hypothesized that the nighttime pre-sleep consumption of LGI CHO would, in the next morning, enhance fat utilization during exercise, decrease GID, and improve 5-km running time compared to a HGI CHO and a placebo (PLA) control.

#### **2. Materials and Methods**

#### *2.1. Subjects*

Trained male (*n* = 8) and female (*n* = 6) endurance runners between the ages of 18 and 45 years were recruited to participate in this study from local running clubs, triathlon teams, by word of mouth, flyers, and through an email distribution around the Skidmore College campus. Participants were included if they met the peak volume of oxygen consumption (VO2peak) qualifications (women: VO2peak <sup>≥</sup> 40 mL·kg−1·min−<sup>1</sup> and men: VO2peak <sup>≥</sup> 45 mL·kg−1·min−1). Menstrual cycle status was recorded for all female participants, though they were scheduled independently of the menstrual cycle phase; notably, 3 out of 6 female participants did not have a menstrual cycle due to hormonal or contraceptive therapy. Participants were excluded if they smoked, had uncontrolled thyroid conditions, had been diagnosed with cardiac or metabolic disorders, regularly consumed anti-inflammatory drugs or any dietary supplements intended to improve performance, or had musculoskeletal injury that limited performance. All experimental procedures and risks of participation were explained verbally and in writing prior to participants providing written informed consent. Approval for this study was granted by the Human Subjects Institutional Review Board (IRB# 1901-786) of Skidmore College and is in accordance with the most recent revisions of the Declaration of Helsinki.

#### *2.2. General Procedures*

This was a double-blinded randomized placebo-controlled study included four total trials: one familiarization trial and three experimental trials. For the experimental trials, participants were randomly assigned to consume either (1) LGI, (2) HGI, or (3) PLA at least 2 h after their last meal and within 30 min prior to sleep on the evening before returning to the laboratory. Participants then arrived to the lab in the morning after an overnight fast (~7–9 h after supplement consumption) for an incremental exercise test (IET) and 5-km time trial (TT) (See Figure 1). Prior to the first experimental trial, participants were required to complete a one-day dietary food and exercise log. Participants were asked to replicate this diet and exercise, as closely as possible, prior to subsequent exercise trials. Participants abstained from the use of non-steroidal anti-inflammatory drugs, caffeine, alcohol, and/or vigorous activity at least 24 h prior to each experimental trial.

**Figure 1.** Experimental Overview; RMR = resting metabolic rate; IET: incremental exercise test.

## *2.3. Supplementation*

Over the span of the study, all participants were randomized to the order in which they received each of the following three supplements: (1) 532 mL of water mixed with 75 g of a HMS (LGI; Orange Flavor, SuperStarch®, The UCAN Co., Woodbridge, CT, USA) (270 kcal; 0 g PRO; 66 g CHO; 0 g FAT), (2) 532 mL of water mixed with 75 g of a HGI glucose-based supplement (Orange flavor, Gatorade®, PepsiCo, Inc., Purchase, NY, USA) (270 kcal; 0 g PRO; 67 g CHO; 0 g FAT), and (3) 532 mL of water mixed with a color and flavor-matched, non-nutritive PLA (PLA; Orange CRUSH flavor packet and Benefiber), with a volume of the powder visually similar to the other experimental conditions. Beverages were of

similar taste, appearance, and consistency. The supplements were pre-packaged in inconspicuously labeled/coded opaque containers by a researcher not otherwise involved in the study.

#### *2.4. Familiarization Protocol*

Participants filled out a physical activity readiness questionnaire (PAR-Q), ACSM health preparticipation screening questionnaire, and a menstrual cycle history form (females only). Height was measured using a stadiometer (Seca 213, portable stadiometer, Chino, CA, USA), while body composition and weight were measured using air displacement plethysmography (BOD POD; COSMED, Chicago, IL, USA) [29].

Peak volume of oxygen consumption (VO2peak) testing was performed to assess baseline cardiorespiratory fitness and inclusion in the study. Gas exchange and ventilatory parameters were measured with a metabolic cart system (TrueOne 2400 Parvomedics, Salt Lake City, UT, USA) [30]. For each individual trial, the metabolic system was calibrated by a flow-calibration with a 3-L calibration syringe and gas analyzer calibration with gas mixture of known concentrations of oxygen (O2) and carbon dioxide (CO2) (16% O2; 4% CO2) according to manufacturer specifications, in addition to environmental data for standardization purposes. Participants were fitted with a nose clip, two-way non-rebreathe valve, and mouthpiece which was supported by a headpiece in order to collect expiratory gases for analysis by the metabolic cart. The VO2peak protocol was performed on a treadmill (Woodway PPS Med, Waukesha, WI) and the protocol required a self-selected constant pace that was "comfortable but challenging." Once the appropriate speed was determined, grade was increased at a rate of 2% every two minutes until the participant reached volitional fatigue [28]. During the last 15 s of each stage, HR was measured using a chest worn HR monitor (H7, Polar USA, Lake Success, NY, USA) and RPE was measured on a 1–10 categorical ratio scale.
