Thus, while it is clear that overfeeding for as little as 3–7 days in humans can lead to increased fat mass and impaired glycemic regulation under experimental steady-state conditions ( 1 , 10 , 12 , 15 , 22 , 28 , 34 , 46 ), the effects of overfeeding on postprandial glycemic regulation, specifically regarding glucose fluxes, is not well characterized. Therefore, our aim was to examine the effects of overfeeding, independently of changes in habitual macronutrient composition, on postprandial glucose metabolism. Specifically, we hypothesized that impairments to postprandial suppression of endogenous glucose production (EGP) could occur, given that in humans as few as 5 days of overfeeding has been previously shown to impair EGP suppression during the hyperinsulinemic clamp ( 24 , 27 , 33 , 46 ). Accordingly, we utilized mixed-meal tolerance testing combined with the variable infusion triple-stable isotope glucose tracer approach [i.e., tracer clamp ( 4 , 32 , 45 )] to assess postprandial glucose fluxes (glucose appearance, production and disposal) following both short-term (5 days) and chronic (28 days) overfeeding in healthy lean young adult males.

Additionally, many human studies investigating glucose metabolism following periods of overfeeding utilize experimental techniques such as the hyperinsulinemic clamp ( 1 , 10 , 12 , 15 , 28 , 35 , 38 ) or intravenous glucose tolerance test (IVGTT), which are not truly representative of postprandial conditions ( 10 ). Importantly, those studies suggesting that rapid induction of hepatic defects in humans occur within 7 days of overfeeding have exclusively utilized steady-state techniques ( 10 , 15 , 40 , 41 ). Although some human overfeeding studies have utilized glucose or mixed-meal tolerance tests to assess glycemic control ( 14 , 22 , 34 ), to the best of our knowledge none have concomitantly utilized glucose isotope tracers to determine glucose fluxes under these conditions.

First, many human overfeeding trials use experimental diets that not only provide energy excess but also alter the composition of macronutrients, favoring an increase in the amount of energy derived from fat ( 10 , 12 , 22 , 34 , 35 , 38 ). Indeed, many of the recent human studies reporting that overfeeding alters glucose metabolism have also provided a high proportion of fat, often greater than 40% of energy intake ( 10 , 12 , 22 , 34 , 38 , 39 ). Therefore, it is not clear whether the observed impairments in glucoregulation are a transient pathological result of the high fat content of the diets, an increase in total energy intake, or simply due to the increased dietary fat availability. Furthermore, it is unlikely that overfeeding one specific macronutrient (i.e., fat) is actually representative of a real-life overeating paradigm. The typical fat composition of diets in most Western countries is ~30–35% energy ( 3 , 32a , 48 ), and it is likely that consumption of energy-dense foods provides excesses of carbohydrate and protein, along with fat.

Short-term experimental overfeeding is a model often used in animal studies to replicate overconsumption in humans, and these studies have consistently demonstrated that defects in hepatic glucose metabolism occur within a few (1–7) days of the onset of overconsumption, preceding the induction of peripheral defects that take several weeks to emerge ( 24 , 27 , 33 , 46 ). Although previous human studies have also observed a similar temporal pattern of glucoregulatory defects, such as rapid development of impaired hepatic glucose metabolism in response to overfeeding ( 10 , 15 , 40 , 41 ), our current understanding of the physiological implications of overfeeding in humans is complicated by a number of factors.

The past few decades have seen an immense rise in both obesity and type 2 diabetes ( 18 ). Considering their rapid worldwide development, it is unlikely that this has been driven by genetics alone. Rather, it is evident that lifestyle factors, such as the broad availability of inexpensive, highly palatable energy-dense foods are playing a significant role in this epidemic ( 15 , 49 ). This widespread overconsumption of energy-dense foods, particularly in the form of processed starch, sugar, and fat ( 8 , 49 ), is characteristic of the Westernized diet and is likely to be a crucial factor leading to the development of insulin resistance and glucose intolerance ( 10 ). Considering that overfeeding in humans, even in the short term (1–7 days), can impair glycemic control and insulin action ( 14 , 22 , 28 , 34 , 35 , 38 ), understanding the processes governing these overfeeding-induced changes may help provide insight into metabolic disease progression.

A priori power calculations were undertaken for the primary outcome measure EGP, as well as R d . Due to the small number of triple-tracer studies in humans assessing lifestyle interventions, the calculations were based on previous rodent studies demonstrating that hepatic and peripheral insulin sensitivity can decrease by 20–40% following overfeeding ( 27 , 33 , 46 ) and previously published human triple-tracer data for typical EGP and R d values ( 32 ). To detect a conservative 25% decrease in total EGP suppression (1,747 ± 306) with 80% power, a sample size of 8 was required. To detect a conservative 20% decrease in glucose R d AUC (6,923 ± 806) with 80% power, a sample size of 6 was required.

Differences between baseline, short-term overfeeding, and chronic overfeeding were assessed with either a paired t -test or one-way or two-way repeated-measures ANOVA. Bonferroni post hoc analysis was used to examine time course differences between the conditions (i.e., baseline vs. short-term overfeeding; baseline vs. chronic overfeeding). All statistical analyses were performed using GraphPad Prism (v. 6.0; La Jolla, CA). All data are presented as means ± SE. Significance was set at P < 0.05.

Glucose fluxes, meal rate of appearance (R a ), rate of disposal (R d ), and EGP were calculated using Steele’s non-steady-state model ( 43 ) as described in detail previously ( 32 ). Insulin secretion rate was calculated using glucose and C-peptide kinetics in a computerized program implementing a regularization method of deconvolution ( 20 ). Hepatic insulin extraction was calculated as insulin secretion rate area under the curve (AUC)/plasma insulin AUC ( 9 ). The AUCs for glucose, insulin, C-peptide, NEFA, TAG, and insulin secretion rate were calculated using the trapezoidal method. Fasting glucose, R d , and EGP are reported as the average of time points −150, −60, −30, −20, −10, and 0 min. Fasting insulin and C-peptide are reported as the average of time points −150 and 0 min.

Samples were injected using a 1:20 split ratio onto a HP-5MS 5% phenylmethylsiloxane column (30.0 m × 250 μm × 0.25 μm; Agilent technologies, Santa Clara, CA) connected to an Agilent 7890B gas chromatograph. Target compounds were detected using an Agilent 5977B mass selective detector (MSD; Agilent Technologies, Santa Clara, CA). The GC program consisted of a 35°C/min ramp starting at 60°C. A final temperature of 280°C was then held for 3 min. Helium was used as the carrier and methane as the reagent gas. The MSD was operated in the selected ion monitoring mode measuring the intact (C1–C6) molecular ions at mass to charge ratios ( m / z ; M0–M+6) 384, 385, 386, 387, 388, 389, and 390, corresponding to natural unlabeled (384, M0), [1- 13 C]- (385, M+1), [6,6- 2 H]- (386, M+2), and [U- 13 C] (390, M+6) glucose. Ion abundances were quantified using the Mass Hunter Workstation (Agilent Technologies). The raw isotopomer data were corrected for natural isotopic background abundance skew using the matrix method ( 30 ), permitting enrichments to be expressed as mole percent excess.

Tracer enrichment in plasma, infusates, and labeled meal samples was measured using methane-positive chemical ionization gas chromatography-mass spectrometry (GC-MS). Preparation of the glucose aldonitrile pentapropionate derivative was undertaken as described by Antoniewicz et al. ( 2 ). Briefly, 10 μl of plasma was mixed with 100 μl of ice-cold analytic-grade methanol and centrifuged for 5 min to precipitate plasma protein. The supernatant (~90 μl) was removed and evaporated to dryness in glass GC inserts under vacuum at 40°C using a centrifugal speed evaporator. Dried samples were dissolved in 50 μl of hydroxylamine hydrochloride solution (20 mg/ml in pyridine) and heated at 90°C for 60 min, after which 100 μl of propionic anhydride was added. Following 30-min incubation at 60°C, samples were dried at 40°C under vacuum as described above and dissolved in 100 μl of ethyl acetate for subsequent analysis via GC-MS.

Participants were required to complete daily checklists during the overfeeding period indicating which snacks were consumed and to complete 3-day diet diaries three times throughout the trial, from days −3 to 0, 2 to 5, and 25 to 28 , as well as Stanford 7-day activity recalls at baseline and after 28 days of overfeeding, according to previously published guidelines ( 31 ). Participants visited the laboratory weekly to monitor bodyweight and return food diaries and for dispensation of snacks and review of checklists so that any deviations from the protocol were quickly identified and corrected. At the end of the study, diets were analyzed for macronutrient composition by using Foodworks 2007, based on the Australian foods database (Xyris Software, QLD, Australia).

All participants completed 28 days of overfeeding. During this period, participants were instructed to consume their regular diets and were provided with snacks to achieve an energy intake of ~5,000 kJ/day above their baseline energy requirements. The overfeeding diet was designed to maintain the macronutrient composition at ~53% carbohydrate, 32% fat, and 15% protein, to be representative of a typical Australian dietary macronutrient composition ( 3 ). The snacks included energy-dense foods such as chips, chocolate, and meal replacement shakes. For the 24 h preceding the short-term and chronic overfeeding mixed-meal tolerance testing trials, all foods were provided at baseline energy requirement plus an additional ~5,000 kJ/day, with a nutrient composition of 55% carbohydrate, 30% fat, and 15% protein.

At the designated time point of 0 min, participants ingested a mixed meal (10 kJ/kg, 45% carbohydrate, 20% protein, and 35% fat) consisting of eggs, cheese, and 1.2 g/kg glucose (including [6,6- 2 H]glucose at an enrichment of 4% wt/vol) in sugar-free Jell-O (Aeroplane Jelly, Victoria, Australia) as the sole carbohydrate source. The meal was consumed within 10 min. At 0 min (i.e., with the first bite), the infusion of [1- 13 C]glucose was altered in a pattern so as to approximate the anticipated fall in EGP (0–10 min = 70%, 10–20 min = 60%, 20–30 min = 50%, 30–180 min = 35%, 180–210 min = 40%, 210–270 min = 50%; percent basal rate of 0.333 µmol·kg −1 ·min −1 ). At the same time, an infusion of [U- 13 C]glucose was started (1.11 µmol·kg −1 ·min −1 ), and the rate was varied to mimic the anticipated appearance of [6,6- 2 H]glucose from the meal (0–10 min = 25%, 10–30 min = 100%, 30–70 min = 65%, 70–90 min = 55%, 90–120 min = 45%, 120–150 min = 35%, 150–180 min = 25%, 180–210 min = 20%, 210–270 min = 10%; percent maximal infusion rate of 1.11 µmol·kg −1 ·min −1 ). Blood samples were taken at 10, 20, 30, 40, 50, 70, 90, 120, 150, 180, 210, and 270 min after meal ingestion.

Experimental trials occurred as we have previously detailed ( 32 ). Briefly, a primed, continuous intravenous infusion of [1- 13 C]glucose was initiated and continued until the end of the study, where a bolus of 33.3 µmol/kg was infused over 5 min followed by a constant infusion (0.333 µmol·kg −1 ·min −1 ) for the following 150 min. Blood samples were taken during the equilibration period at designated time points: −150 (immediately before infusion), −60, −30, −20, −10, and 0 min. All basal and postprandial blood samples were immediately spun in a centrifuge at 3,000 g for 15 min at 4°C, and plasma was stored at −80°C until analysis.

This study was conducted according to the Declaration of Helsinki, all procedures were approved by the Deakin University Human Research Ethics Committee, and all participants provided written informed consent. Participants arrived at the clinical research facility at 0700 in the overnight (10-h)-fasted state, having refrained from exercise and alcohol consumption for 48 h. For the 24 h before the baseline experimental trial, participants were provided with an energy maintenance diet (9,783 kJ, 55% carbohydrate, 30% fat, 15% protein) designed to be representative of the typical macronutrient composition of an Australian diet ( 40 ). A 22-gauge cannula was inserted into a vein of each forearm for tracer infusions and venous blood sampling, respectively. Sterile stable isotopes; [1- 13 C]glucose, [6,6- 2 H]glucose, and [U- 13 C]glucose (Cambridge Isotope Laboratories, Andover, MA) were prepared as previously described ( 32 ).

Following short-term overfeeding, plasma meal-derived glucose concentration was modestly but significantly higher compared with baseline at 20 and 30 min (diet × time interaction; Fig. 5 A ), although this did not translate into a significant change to the total integrated (689 ± 84 vs. 683 ± 66 mmol/l) or 0- to 120-min meal glucose AUC (293 ± 27 vs. 333 ± 30 mmol/l). However, with regard to chronic overfeeding, meal-derived glucose concentration was significantly higher compared with baseline at 20, 40, 70, 90, and 120 min (diet × time interaction; Fig. 5 C ), and this was associated with a significant increase to the total integrated (689 ± 84 vs. 790 ± 73 mmol/l, P < 0.05, baseline vs. chronic overfeeding, respectively), and 0- to 120-min integrated meal glucose AUC (293 ± 27 vs. 394 ± 30 mmol/l, P < 0.05). The concentration of endogenous glucose was not significantly altered by short-term or chronic overfeeding ( Fig. 5 , B and D ).

Chronic overfeeding did not alter fasting EGP or R d , nor the postprandial suppression of EGP compared with baseline ( Fig. 4 F ). Compared with baseline, both meal glucose R a and glucose R d were significantly increased following chronic overfeeding at 20 and 30 min ( Fig. 4 , D and E ). While the 0- to 120-min R d (4,209 ± 337 vs. 4,785 ± 305 µmol/kg, P < 0.05, baseline vs. chronic overfeeding, respectively) and R a (3,450 ±315 vs. 4,027 ± 289 µmol/kg, P < 0.05) AUC was increased by chronic overfeeding, the total postprandial AUC for R d and R a was unaltered.

Short-term overfeeding significantly increased fasting rates of EGP (10.9 ± 0.8 vs. 11.5 ± 0.8 μmol·kg −1 ·min −1 , P < 0.05, baseline vs. short-term overfeeding, respectively; Fig. 4 C ) and glucose R d (11.2 ± 0.8 vs. 11.9 ± 0.8 μmol·kg −1 ·min −1 , P < 0.05, baseline vs. short-term overfeeding, respectively; Fig. 4 B ). Although postprandial EGP suppression was unaltered by short-term overfeeding ( Fig. 4 C ), both meal glucose R a and glucose R d were significantly increased over the initial 90 min of the postprandial period ( Fig. 4 , A and B ). Consequently, there was a significant increase in both the total R a AUC (5,724 ± 334 vs. 6,382 ±271 µmol/kg, P < 0.05, baseline vs. short-term overfeeding, respectively) and 0- to 120-min R a AUC (3,450 ± 315 vs. 4321 ± 364 µmol/kg, P < 0.05), as well as the total R d AUC (7,328 ± 426 vs. 8,036 ± 388 µmol/kg, P < 0.05) and 0- to 120-min R d AUC (4,209 ± 0.337 vs. 5,057 ± 391 µmol/kg, P < 0.05).

With regard to chronic overfeeding, the fasting glucose, insulin, C-peptide, NEFA, and TAG were unaltered compared with baseline ( Table 2 ). However, chronic overfeeding significantly increased the integrated postprandial glucose AUC from 0 to 120 min ( Table 2 ) but not the total AUC for glucose. Additionally, the postprandial insulin AUC from 0 to 120 min was significantly increased ( Table 2 ), while the pattern also differed such that there was a biphasic response following chronic overfeeding. Chronic overfeeding also significantly increased plasma C-peptide levels at 90 min ( Fig. 2 C ) but did not significantly alter the C-peptide AUC ( Table 2 ). Both insulin secretion rate ( Fig. 2 D ) and insulin clearance (0.163 ±0.023 vs. 0.138 ± 0.020 l·min −1 ·m −2 , baseline vs. chronic overfeeding, respectively, P = 0.179) were not significantly altered by chronic overfeeding. Postprandial NEFA ( Fig. 2 E ) and TAG (data not shown) were not altered by chronic overfeeding.

Fasting glucose, C-peptide, NEFA, and TAG were unaltered by short-term overfeeding, although fasting insulin was significantly increased compared with baseline ( Table 2 ). Compared with the glycemic responses at baseline, short-term overfeeding did not result in a significant change to the postprandial glucose excursion ( Fig. 1 A ). Although short-term overfeeding significantly increased plasma insulin and C-peptide levels at 30 min compared with baseline (diet × time interaction, P < 0.05; Fig. 1 , B and C ), this did not result in a significant alteration to the postprandial insulin or C-peptide AUC ( Table 2 ). Additionally, although insulin secretion rate ( Fig. 1 D ) was unaltered, insulin clearance tended to decrease ( P = 0.060) following short-term overfeeding (0.163 ± 0.02 vs. 0.128 ± 0.0.01 l·min −1 ·m −2 ; baseline vs. short-term overfeeding, respectively). Postprandial NEFA ( Fig. 1 E ) and TAG (data not shown) were not altered by acute overfeeding.

Participants consumed 4,938 ±87 kJ of additional energy to that normally supplied by their regular diet, which equated to an additional 46% total energy intake ( Table 1 ). Participants achieved 96 and 98% compliance for consuming provided overfeeding snacks, for 5 and 28 days, respectively. Dietary energy and macronutrient content derived from the participants’ normal diet was not significantly altered from baseline. Total dietary fat, carbohydrate, and protein intake significantly increased by overfeeding, whereas the percentage of energy derived from these macronutrients did not change. Whereas short-term overfeeding had little effect on body composition ( Table 1 ), body mass and fat mass were significantly higher after 28 days of overfeeding than at baseline (1.64 ± 0.40 kg, P < 0.05; 1.32 ± 0.18 kg, P < 0.05, respectively; Table 1 ). Both short-term and chronic overfeeding significantly increased visceral fat volume by 59.5 ± 2.0 and 70.1 ± 2.7 g/cm 2 , respectively ( P < 0.05; Table 1 ).

DISCUSSION

Our findings show that both short-term (5 days) and chronic (28 days) overfeeding in healthy young males, independently of changes in macronutrient composition, elicit only modest alterations to body composition, glycemia, and insulinemia and, in direct contrast to our hypothesis, no change to the pattern and magnitude of postprandial EGP suppression. However, both short-term and chronic overfeeding significantly increased meal glucose R a , and although glucose R d closely matched this rise in R a , only chronic overfeeding demonstrated a significant, albeit small, increase in postprandial glycemia. Interestingly, the change in plasma insulin occurred despite there being no change in insulin secretion rate, suggesting that the modest increase in glycemic and insulinemic excursions following chronic overfeeding likely permits more efficient stimulation of glucose flux without the need to drive a large change in compensatory β-cell insulin secretion.

An important finding of the present study was that fasting glucose was unaltered by overfeeding, and postprandial glycemia was only modestly increased by chronic, but unchanged by short-term, overfeeding. This is in contrast to a range of previous data in humans demonstrating that overfeeding for 3–7 days increases fasting (10, 12, 13, 22, 34) and postprandial (22, 34) glycemia in healthy humans. However, the majority of these overfeeding studies utilized diets that substantially increased the relative amount of energy derived from fat (10, 12, 22, 34). Indeed, following only 7 days of overfeeding a diet containing 60% energy from fat (increased from 31.5% in the habitual diet), Parry et al. (34) recently demonstrated similar increases in postprandial glucose AUC and insulin AUC during a meal tolerance test as in the current chronic overfeeding study. Thus, 7 days of high-fat overfeeding has a similar impact on postprandial glucose and insulin as 28 days of overfeeding with a mixed macronutrient composition. Although some studies utilizing habitual macronutrient compositions or high-carbohydrate overfeeding diets have demonstrated significant alterations to fasting or postprandial glycemia in the short term (13), the changes are typically much smaller than those from diets utilizing a high fat composition (1, 28). Thus, overfeeding with a diet that increases the proportion of dietary fat may bias toward an impairment in glucoregulatory function by increasing reliance on fat metabolism at the expense of carbohydrate metabolism, more rapidly altering glucoregulatory function.

The macronutrient composition of overfeeding is also a key consideration in regard to alterations to body composition. The relatively modest 1.2-kg increase in fat mass after 28 days of overfeeding in the current study compared with previous studies is likely a function of the habitual macronutrient composition of the study diet. Indeed, previous studies (19, 29) have demonstrated that 2–3 wk of overfeeding with high-fat diets produces significantly greater adipose tissue accumulation compared with overfeeding with carbohydrate-based diets. This heterogeneity in fat storage responses between fat- and carbohydrate-based diets was shown to occur as a result of progressive increases in total energy expenditure and carbohydrate oxidation following high-carbohydrate overfeeding (19). Thus, greater carbohydrate oxidation offsets the increase in energy intake and minimizes nutrient storage compared with a high-fat diet (19), highlighting the potent metabolic effect of consuming a high-fat diet above that of energy excess with an alternate macronutrient composition.

Increases in total body fat are strongly linked with metabolic disease progression (21), and this is especially true for increases in visceral fat (17, 47). Interestingly, in the current study, visceral adipose volume was increased after both short-term and chronic overfeeding despite there being no change in body weight or fat mass after 5 days. However, despite the similar change to visceral adipose volume after 5 and 28 days of overfeeding, the postprandial glycemic response was increased only following chronic overfeeding. On the other hand, Knudsen et al. (25) recently demonstrated that decreased insulin sensitivity as assessed by the Matsuda index and euglycemic-hyperinsulinemic clamp, and increased insulin during an oral glucose tolerance test occurred in response to combined overfeeding and inactivity before any change to visceral adipose tissue volume. Taken together, these findings suggest that the initial steps in the development of disturbed glucoregulatory function are not necessarily linked to visceral fat accumulation (25, 44).

In addition to increased postprandial glycemia, the systemic insulin response was increased following chronic overfeeding, suggesting some degree of insulin resistance. Modest changes in C-peptide during the meal tolerance test suggest that the accentuated insulin response following chronic overfeeding may be related to changes to insulin secretion. However, changes to insulin secretion also appear to be at best minimal, since the pattern and integrated response of modeled insulin secretion was not altered following overfeeding. Additionally, although modeled insulin clearance was not significantly altered following chronic overfeeding, it is possible that the current study was underpowered to detect modest changes in model-derived variables. Considering that insulin clearance was significantly decreased by ~20% following acute overfeeding, this suggests that decreased insulin clearance may occur in response to overfeeding as a mechanism to allow an appropriate degree of insulin into the periphery without placing an additional burden on the β-cell. In reality, a combination of modest changes to both secretion and clearance likely explain the altered insulin response, as previous studies have shown that significant changes in body weight and insulin resistance increase both the postprandial insulin secretion and the insulin clearance rate to maintain glucose homeostasis in overweight and obese subjects (37).

With regard to postprandial glucose fluxes, it is possible that the increased postprandial glycemia following chronic overfeeding occurred as a mechanism for glucose itself to stimulate R d , considering that the mass effect of glucose to stimulate its own uptake and suppress its own production is a determinant of glucose tolerance (6, 26). Peterson et al. (35) recently demonstrated that the early stages of overfeeding-induced alterations to glucoregulatory function appear to be driven more by declines in nonoxidative rather than oxidative glucose metabolism, and it can be hypothesized that hyperglycemia may serve to compensate for the defects in nonoxidative disposal. Furthermore, the need to stimulate increased glucose R d following both short-term and chronic overfeeding occurred in response to an increase in the systemic meal glucose R a , suggesting that overfeeding may, at least transiently, lead to an adaptation in splanchnic tissues. Although speculative, the possible mechanisms that may serve to explain the increased meal glucose R a in response to overfeeding include increased gastric emptying and/or reduced hepatic extraction of glucose. Considering that the rate of gastric emptying has a direct effect on the rate of glucose appearance after a meal (11), and that gastric emptying is accelerated early in the development of type 2 diabetes (16), this offers a possible explanation for the increased meal Ra following short-term and chronic overfeeding in the current study.

The observed increase in fasting rates of EGP in response to short-term overfeeding in the current study are consistent with previous findings in humans that have observed that development of impaired hepatic glucose metabolism occurs rapidly in response to overfeeding (10, 15, 40, 41). However, in contrast to this prevailing view, neither short-term nor chronic overfeeding was associated with a reduction in the postprandial suppression of EGP, and it is important to consider that previous studies have utilized steady-state measures of EGP during fasting (10, 13, 40) or hyperinsulinemic clamp (15) conditions. Under postprandial, non-steady-state conditions, rates of glucose flux are governed by the integrated regulation of β-cell insulin secretion, insulin sensitivity, and glucose effectiveness (26). A number of researchers have demonstrated that effective compensation by these postprandial regulatory mechanisms can normalize postprandial suppression of EGP to maintain normal postprandial glucose concentrations in the face of insulin resistance (5, 7, 23). Thus, despite increased fasting rates of EGP in response to short-term overfeeding in the current study, postprandial suppression of EGP was not reduced, likely due to effective compensation by portal vein hyperinsulinemia and hyperglycemia.

Another important observation in the present study is that fasting glucose was unchanged after 5 days of overfeeding despite increased fasting EGP. This occurred concomitantly with an increase in fasting insulin, suggesting the development of hepatic insulin resistance, although fasting R d was also increased. Schwarz et al. (40) also observed increased fasting EGP in response to 5 days of carbohydrate overfeeding, although this also induced secondary effects in regard to increasing insulin secretion and suppressing lipolysis (40), suggesting that increased fasting EGP may be an adaptation that shifts whole body fuel selection toward glucose in response to dietary carbohydrate surplus. Indeed, another previous study utilizing high-carbohydrate overfeeding (1) observed an increase in fasting insulin, as in the current study, but no change to insulin sensitivity as determined during the clamp, despite an increase in insulin signaling. This suggests that early adaptations in response to carbohydrate overfeeding are directed at increasing glucose disposal to maintain whole body insulin sensitivity. Thus, in the present study, alterations to fasting EGP, R d , and insulin after 5 days of overfeeding likely represent a physiological adaptation to short-term energy excess to support a shift in whole body metabolism toward increased carbohydrate oxidation.