Alternate day calorie restriction improves clinical findings and reduces
markers of oxidative stress and inflammation in overweight adults
with moderate asthma
James B. Johnsona,⁎, Warren Summerb, Roy G. Cutlerc, Bronwen Martinc, Dong-Hoon Hyunc,
Vishwa D. Dixitd, Michelle Pearsonc, Matthew Nassarc, Richard Tellejohanc, Stuart Maudsleyc,
Olga Carlsone, Sujit Johnf, Donald R. Laubg, Mark P. Mattsonc
aDepartment of Surgery, Louisiana State University Medical Center, New Orleans, LA 70006, USA
bDepartment of Pulmonary Medicine, Louisiana State University Medical Center, New Orleans, LA, USA
cLaboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD, USA
dLaboratory of Immunology, National Institute on Aging Intramural Research Program, Baltimore, MD, USA
eDiabetes Section, National Institute on Aging Intramural Research Program, Baltimore, MD, USA
fDepartment of Statistics, Stanford University, Stanford, CA, USA
gDepartment of Surgery, Stanford University, Palo Alto, CA, USA
Received 11 August 2006; revised 5 November 2006; accepted 8 December 2006
Available online 14 December 2006
Asthma is an increasingly common disorder responsible for considerable morbidity and mortality. Although obesity is a risk factor for asthma
and weight loss can improve symptoms, many patients do not adhere to low calorie diets and the impact of dietary restriction on the disease
process is unknown. A study was designed to determine if overweight asthma patients would adhere to an alternate day calorie restriction (ADCR)
dietary regimen, and to establish the effects of the diet on their symptoms, pulmonary function and markers of oxidative stress, and inflammation.
Ten subjects with BMI>30 were maintained for 8 weeks on a dietary regimen in which they ate ad libitum every other day, while consuming less
than 20% of their normal calorie intake on the intervening days. At baseline, and at designated time points during the 8-week study, asthma
control, symptoms, and Quality of Life questionnaires (ACQ, ASUI, mini-AQLQ) were assessed and blood was collected for analyses of markers
of general health, oxidative stress, and inflammation. Peak expiratory flow (PEF) was measured daily on awakening. Pre-and postbronchodilator
spirometry was obtained at baseline and 8 weeks. Nine of the subjects adhered to the diet and lost an average of 8% of their initial weight during
the study. Their asthma-related symptoms, control, and QOL improved significantly, and PEF increased significantly, within 2 weeks of diet
initiation; these changes persisted for the duration of the study. Spirometery was unaffected by ADCR. Levels of serum β-hydroxybutyrate were
increased and levels of leptin were decreased on CR days, indicating a shift in energy metabolism toward utilization of fatty acids and confirming
compliance with the diet. The improved clinical findings were associated with decreased levels of serum cholesterol and triglycerides, striking
reductions in markers of oxidative stress (8-isoprostane, nitrotyrosine, protein carbonyls, and 4-hydroxynonenal adducts), and increased levels of
the antioxidant uric acid. Indicators of inflammation, including serum tumor necrosis factor-α and brain-derived neurotrophic factor, were also
significantly decreased by ADCR. Compliance with the ADCR diet was high, symptoms and pulmonary function improved, and oxidative stress
and inflammation declined in response to the dietary intervention. These findings demonstrate rapid and sustained beneficial effects of ADCR on
the underlying disease process in subjects with asthma, suggesting a novel approach for therapeutic intervention in this disorder.
© 2006 Elsevier Inc. All rights reserved.
Keywords: AQLQ; Isoprostanes; Peak expiratory flow; Protein carbonyls; Nitrotyrosine; BDNF; Spirometry; Tumor necrosis factor; Oxidative stress
Free Radical Biology & Medicine 42 (2007) 665–674
Abbreviations: ACQ, Juniper Asthma Control Questionnaire; ADCR, alternate day calorie restriction; AL, ad libitum; ASUI, Asthma Symptom Utility Index;
BDNF, brain-derived neurotrophic factor; CR, caloric restriction; mini-AQLQ, Juniper mini-Asthma Quality of Life Questionnaire; PEF, peak expiratory flow; TNF,
tumor necrosis factor.
E-mail address: firstname.lastname@example.org (J.B. Johnson).
0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
The cause(s) and pathogenic mechanisms of asthma are
poorly understood, and available treatments can alleviate
symptoms but do not reverse the disease process . The
prevalence of asthma in industrialized countries throughout the
world has increased significantly during the past 30 years,
of increases in specific allergens. Instead, increasing evidence
points to a link between overeating/obesity and asthma. Weight
loss often improves asthma symptoms in obese subjects , and
low calorie diets and exercise programs result in weight loss and
can reduce asthma symptoms in overweight children and adults
[4,5]. However, while obesity is a risk factor for asthma-related
symptoms such as wheezing, it may not be a cause of airway
loss modifies the asthma disease process.
The molecular and cellular mechanisms underlying airway
hyperresponsiveness and asthma symptoms are complex and
poorly understood. Two general alterations in the lungs are
increased oxidative stress and inflammation [7–11]. The local
changes in the lungs are associated with increases in markers of
inflammation and oxidative stress in the blood including TNF
, interleukin-6  and lipid peroxidation products . In
addition, circulating levels of brain-derived neurotrophic factor
(BDNF) are increased in patients with asthma and other allergic
disorders [15,16]. Although capable of transiently relieving
asthma symptoms, agents such as corticosteroids and β-
adrenoreceptor agonists do not block or reverse the underlying
disease process and their long-term use poses a considerable
risk of morbidity and mortality [17,18].
Caloric restriction (CR) improves numerous health indi-
cators in rodents, monkeys, and humans, including those
associated with risk of cardiovascular disease, type 2 diabetes,
and cancers [19–21]. Similar to daily CR (on a long-term
basis), intermittent CR can extend lifespan and protect multiple
organ systems against disease in rodents [22–24]. However,
despite considerable evidence that intermittent CR is beneficial
in rodent disease models, the potential application of
intermittent CR to human diseases is largely untested . In
light of the poor adherence of subjects to continuous CR diets
and adverse consequences associated with gastric bypass
surgery and pharmacological interventions , we designed
a pilot study aimed at determining the feasibility and efficacy
of an intermittent CR diet in treating overweight patients with
This study was approved by an independent Review Board
(Cresent City IRB) and analyses of serum samples were
approved by the IRB of the National Institute on Aging
Intramural Research Program. Participants were recruited
through newspaper advertisements in the New Orleans metro-
politan area. Inclusion and exclusion criteria were assessed by
telephone, an in person interview, and a physician-conducted
examination. Participants meeting the following criteria were
included in the study: stable body weight with BMI>30 and
less than 300 pounds; prior diagnosis of stable moderate
persistent asthma as defined by the “Expert Panel Report 2”
(NHLBI) ; FEV1or peak expiratory flow (PEF) >50%;
daily symptoms with use of inhaled short-acting beta2-agonist
and controller, medication regimen stable for at least 30 days
prior to the screening visit; medical history provided by the
subject or the subject's physician did not indicate any
potential risk to the subject as the result of the study. The
subjects were in general good health based on assessment by
the investigators and willing to follow instructions and
complete study procedures as required by the protocol. All
subjects had demonstrated a >12% postbronchodilator
increase in FEV1 documented in the past 2 years. Subjects
were excluded if they had a history of smoking, were taking
systemic corticosteroids within the prior 6 weeks, were using
hypoglycemic agents or insulin at screening, or if it was felt
such medication might be needed during the study. The
dosages of all medications, including over the counter,
herbals, and dietary supplements were recorded.
Ten subjects (8 females and 2 males) with inactive
lifestyles and stable moderate persistent asthma with daily
symptoms were enrolled in the study as a single cohort. The
experimental design involved evaluation of clinical and
biochemical variables in subjects at baseline and at designated
time points during the course of a 2-month alternate day CR
(ADCR) dietary regimen. In this longitudinal design, the
baseline value for each subject served as the control value for
that subject to which ADCR diet values were compared. After
a 14-day prediet period during which baseline variables were
recorded, all subjects initiated ADCR in which women were
instructed to consume 320 calories and men 380 calories of a
commercially available canned meal replacement shake
(Atkins Advantage or Carb Solutions) provided to the
subjects. On the other day subjects ate ad libitum (AL).
Diary cards and instructions were given to the subjects during
the 14-day baseline period. On the last day of the baseline
period subjects returned their diary cards and were given new
cards and instructions on how to follow the diet, including the
number of calories to be consumed on each CR day. They
were told to eat on the AL day whatever they normally ate
and to the point of satisfaction but not to intentionally overeat.
The subjects were told to continue taking the vitamins and
herbal supplements they were taking prior to the study. The
principal investigator and ancillary personnel met each week
with all the participants for 1 h in the evening to provide
group support. Topics of discussion were limited to subjects'
reaction to the dietary pattern. Subjects were weighed on Days
1, 15, 29, and 57 using a calibrated balance scale. Blood
draws were taken at baseline and on consecutive AL and CR
days at the 2, 4, and 8-week time points.
666J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
Evaluation of asthma symptoms and pulmonary function
Three different questionnaires were used. The Juniper mini-
Asthma Quality of Life Questionnaire (mini-AQLQ) and the
Juniper Asthma Control Questionnaire (ACQ) were completed
at baseline and end of study. The Asthma Symptom Utility
Index (ASUI) was completed at baseline and every 2 weeks.
The mini-AQLQ has four domains: symptoms, activity
limitations, emotional function, and environmental stimuli.
The ASUI has five domains, all of which are symptoms:
cough, wheeze, dyspnea, sleeplessness, and medication side
effects. The ACQ has six domains and spirometry. It measures
degree of control of the disease, mainly with questions related
to symptoms. Thus, the mini-AQLQ measures perceived QOL
improvement and emotional response, whereas the ASUI and
ACQ measure primarily symptoms. Scores for the mini-
AQLQ and ACQ were analyzed using the package provided
by Dr. Juniper. The ASUI was scored according to published
methods . Participants were trained in the use of the peak
flow meter (mini-Wright by Ferraris). The best of three PEF
measurements were recorded on awakening, during a 14-day
baseline period, and daily during the 58-day study period.
Spirometry before and after albuterol was performed during
baseline and at 8 weeks by a certified respiratory therapist
using the Schilling spirometer (Model: Type SP-1) under the
supervision of the pulmonologist. The best of three attempts
was recorded before and after albuterol during baseline and at
the end of the study.
Assessments of hunger and mood
A hunger/ mood/energy scale was created for this study
because of anecdotal reporting by previous patients of improved
mood and energy levels when on a similar diet, and the lack of a
mood/energy level measure in existing asthma or psychological
questionnaires. Subjects recorded the level of hunger and mood/
energy for each 2-h segment daily during baseline and
throughout the study. The hunger scale ranged from 1 to 10
and 10 being “extremely hungry, never been hungrier.” The
mood/energy scale ranged from 1 to 10 with 1 being “lowest
energy level ever” and 10 being “highest energy level ever.”
Analyses of serum samples
Fasting blood samples were drawn after an overnight fast
on consecutive AL and CR days (Days 1, 2, 15, 16, 29, 30,
57, and 58). Samples taken on consecutive CR and AL days
were analyzed in order to determine whether the variables
being measured changed daily in response to the ADCR
regimen. Most variables changed progressively with increas-
ing time on the ADCR diet, but did not change acutely
between consecutive AL and CR days. Serum lipids, insulin,
glucose, and C-reactive protein were measured in the clinical
laboratory (Quest Diagnostics, New Orleans, LA) using
standard methods in samples drawn on Days 1, 15, 29, and
57 (only Days 1 and 57 were used for statistical analysis).
Serum TNFα, BDNF, protein carbonyls, nitrotyrosine, 8-
isoprostane, 4-hydroxynonenal adducts, and ceramides were
measured in samples drawn on Days 1, 2, 15, 16, 29, 30, 57,
and 58. TNFα levels were measured using a commercially
available ultrasensitive ELISA kit (Biosource Int., Camarillo,
CA). Serum BDNF concentrations were measured using a
commercially available ELISA kit (Promega, San Luis
Obispo, CA). Levels of protein carbonyls, nitrotyrosine, and
8-isoprostane were quantified using methods described
previously [29,30]. Levels of lysine and histidine adducts of
4-hydroxynonenal and long-chain ceramides were measured
by tandem mass spectrometry methods described previously
. Serum leptin and ghrelin concentrations were quantified
using ELISA kits from Linco Research Inc. (St. Charles, MO)
and Phoenix Pharmaceuticals (Belmont, CA), respectively.
Concentrations of total ketone bodies (acetoacetate and 3-
hydroxybutyrate) were measured using a Total Ketone Bodies
kit (Catalog Nos. 415-73301 and 411-73401) from Wako
Diagnostics USA (Richmond VA), on a Roche Cobas Fara II
robotic chemical analyzer according to the manufacturers
specifications. The Total Ketone Body calibrator set (Catalog
No. 412-73791) was used to produce the standard curve and
the Total Ketone Body control (Catalog No. 418-73891) was
used to insure accuracy between assay runs. Uric acid was
measured using a Uric Acid kit (Catalog No. 237-60) from
Diagnostic Chemicals Limited (Oxford, CT), on a Roche
Cobas Fara II robotic chemical analyzer according to the
For those measurements that were normally distributed,
paired t tests and Pearson's correlation coefficients were used
for the analyses. Statistical comparisons of variables in serum
samples during the course of the study with the baseline values
were made using ANOVA and either the Student-Newman-
Keuls or Bonferroni post hoc tests. For nonnormal measure-
ments, Wilcoxon signed rank-sum test and Spearman's
correlation coefficients were used. Two-sided tests were used
for all the comparisons and a P value of 0.05 or less was
considered statistically significant and a P value of 0.01 or less
was considered highly statistically significant. All the analyses
were done using SAS version 9.1.
Alternate day calorie restriction improves asthma symptoms
and pulmonary function
Of 40 responders to the newspaper advertisement, 23 met
inclusion and exclusion criteria and 14 agreed to enroll in the
study. Of these, one died of unknown causes during the
baseline, one dropped out due to a change in vacation plans
during baseline, one decided not to continue during the first
study week, and one dropped out the second study week due to
work-related travel. Of the remaining 10, 9 completed the
study; one subject did not complete the study because she
667 J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
volunteered that she was noncompliant with the CR regimen.
Subjects lost an average of 8% (8.5 kg) of their body weight
during the course of the study, confirming their adherence to
the ADCR regimen (Fig. 1a). The perceived mood and energy
of the subjects increased progressively during the first 3 weeks
of the ADCR diet and remained significantly elevated for the
duration of the study (Fig. 1b). Analysis of the hunger rating
scale indicated that the subject's perceived hunger did not
increase significantly over baseline values during the course of
the study (Fig. 1c). There was a significantly higher level of
hunger on CR days compared to the ad libitum days
throughout the study. PEF increased by a highly significant
amount from a baseline level of 335 L/min to a level of 382 L/
min during the first 3 weeks of the ADCR period, and
remained elevated throughout the 8-week study period (Fig.
1d) (P<0.009 at 8 weeks). There were no significant
differences between FEV1 (forced expiratory flow in 1 s)
values at baseline and at 8 weeks (Table 1). However, the
FEV1 after albuterol administration was significantly greater at
8 weeks compared to baseline (Table 1), suggesting that the
ADCR diet resulted in improved bronchial responsiveness.
There was also a highly significant improvement in the ASUI
scores (0.25±0.17 (P<0.002) Table 1; Fig. 2a) which occurred
within 2 weeks and was maintained throughout the 8-week
ADCR diet. The mini-AQLQ scores of the subjects were
significantly higher in all four domains (asthma symptoms,
activity limitations, emotional function, and environmental
stimuli) at the end of the study compared to baseline,
demonstrating a beneficial effect of the ADCR diet on weight
related or on asthma quality of life (Fig. 2b). The overall change
in the mini-AQLQ was 2.1±1.4 units (P<0.004) or 61%.
Similarly, there were significant positive effects of the ADCR
diet on the ACQ score which changed –1.3±0.7 (P<0.0015) or
Effects of ADCR on markers of lipid and energy metabolism in
Body weight reduction in obese subjects is often associated
with decreases in risk factors for cardiovascular disease and
diabetes. We therefore measured concentrations of lipids (total
Fig. 1. Asthma subjects lose weight and exhibit improved mood and peak airflow when maintained on an alternate day calorie restriction diet. Body weights (a),
mood/energy scores (b), hunger scores (c), and peak expiratory flow (d) were measured at baseline and at the indicated time points during the 2-month ADCR
Results of analyses of pulmonary variables
Peak flow (L/min)
FEV1 (predicted after
14.4±4.1 (%) 0.0081
5.3±3.7 (%) 0.2152
10.5±5.1 (%) 0.0156
668J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
cholesterol, LDL cholesterol, HDL cholesterol and triglycer-
ides), C-reactive protein (CRP), glucose, and insulin in serum
samples taken at baseline and after 8 weeks on the ADCR diet.
Levels of total cholesterol and triglycerides were significantly
lower at 8 weeks compared to baseline, while levels of HDL
cholesterol were significantly increased at 8 weeks (Fig. 3a;
Table 2). The ADCR diet had no significant effect on serum
levels of LDL, glucose, insulin, or CRP (Table 2).
The body weight of subjects on the ADCR diet decreased
progressively, suggesting that they were compliant with the diet
throughout the study. To confirm compliance and to provide
insight into the effects of the ADCR diet on energy metabolism
we measured concentrations of ketone bodies (acetoacetate and
3-hydroxybutyrate) in serum samples taken on consecutive ad
libitum and CR days at baseline and at 2, 4, and 8 weeks.
Levels of ketone bodies reliably increase during extended
periods of fasting or caloric restriction . We found that
levels of ketone bodies were elevated 4-to 6-fold on CR days
compared to ad libitum days, consistent with adherence of the
subjects to the diet (Fig. 3b). There was a significant increase in
levels of 3-hydroxybutyrate on ad libitum days at 4 and
8 weeks of the ADCR regimen compared to baseline levels
(Fig. 3b). Levels of circulating leptin increase in the fed state
and suppress appetite, whereas ghrelin levels increase during
fasting and increase appetite . We found that leptin levels
were lower on CR days compared to AL days throughout the
study, and there was a progressive decrease in leptin levels on
AL days during the 8-week diet period (Fig. 3c). In the case of
ghrelin there was a transient increase in levels on the AL day at
the 2-week diet point, but ghrelin levels were not significantly
affected by diet on either AL or CR days at the 4 and 8-week
time points (Fig. 3d). There were no significant differences in
ghrelin levels on AL compared to CR days at baseline and 4
and 8 weeks.
ADCR reduces markers of inflammation and oxidative stress in
The concentration of TNFα in serum was unchanged after
2 weeks on the ADCR diet. However, there was a highly
significant reduction in serum TNFα levels in the CR day
sample at 4 weeks, and in both the ad libitum and the CR
samples at 8 weeks (Fig. 4a). There was a significant decrease
in circulating BDNF levels that occurred within the first
2 weeks of the dietary intervention, decreased further at
4 weeks, and remained low at 8 weeks (Fig. 4b). Ceramides
are liberated from membrane sphingomyelin in response to
inflammatory cytokine receptor activation and oxidative stress
and levels of ceramides are elevated in affected tissues and
body fluids in several inflammatory and infectious diseases
[34–36]. Levels of ceramides C16:0, C18:0, C22:0, and
C24:1 were significantly decreased on both ad libitum and
CR days within 2 weeks of ADCR diet initiation and
remained at the lower levels for the duration of the 8-week
period (Fig. 4c). These reductions in levels of circulating
TNFα, BDNF, and ceramides in response to the ADCR diet
suggest that this dietary intervention reduces inflammation in
Levels of protein carbonyls, a measure of protein
oxidation, decreased significantly on both ad libitum and
CR days within 2 weeks of diet initiation, continued to
decrease through 4 weeks, and remained low through 8 weeks
(Fig. 5a). Progressive and highly significant decreases in
serum levels of nitrotyrosine and 8-isoprostane also occurred
during the course of the 8-week ADCR diet period (Figs. 5b
and c). Levels of histidine and lysine 4-hydroxynonenal
adducts were progressively and significantly decreased during
the course of the 8-week ADCR diet period; levels of these
adducts were decreased on both ad libitum and CR days (Fig.
5d). The magnitude of the decreases in each marker of
oxidative stress was large; at the end of the 8-week study
levels of protein carbonyls and 8-isoprostane were less than
20% of baseline levels, levels of nitrotyrosine were less than
10% of baseline values, and levels of 4-hydroxynonenal
adducts decreased by approximately 50% (Fig. 5). Finally, we
measured levels of uric acid, a major antioxidant scavenger of
hydroxyl radical and peroxynitrite , in serum samples
from the subjects. Uric acid levels increased significantly (by
approximately 20%) within 2 weeks of ADCR diet initiation
Fig. 2. Alternate day calorie restrictionresults in improvedsymptoms in subjects
with asthma. (a) MiniAQLQ scores for four domains (symptoms, activity
limitations, emotional function, and environmental stimuli) in subjects at
baseline and after 8 weeks of ADCR. The differences between the 8-week and
baseline values were significantly different for each of the four domains
(P<0.004). (b) ASUI scores increased rapidly and significantly (P<0.003)
within 2 weeks of diet initiation.
669J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
and remained elevated through 8 weeks (Fig. 6), consistent
with less oxidative stress.
Nine of the 10 asthma subjects who began the ADCR
regimen complied with the diet, as indicated by progressive
weight loss, and completed the study. All 9 subjects exhibited
improved asthma symptoms, control, and quality of life,
demonstrating a clinical benefit of the ADCR diet. An
improvement of ACQ or mini AQLQ score of 0.5 is con-
sidered clinically important and has been repeatedly shown to
be useful in research and management of individual asthma
patients. In a recent clinical study of 1414 asthma patients
newly started on either fluticosone proprionate or montelucast
an improvement in ACQ and mini-AQLQ scores of >1 unit or
47% and 25%, respectively, was observed . Our ADCR
study recorded a 54% improvement in ACQ and 61%
improvement in mini-AQLQ in patients already on baseline
controller therapy. Although medical and surgical-induced
weight loss is also associated with similar degrees of quality of
life improvement (SF-36) our patients also demonstrated
improvement in asthma specific control (ACQ) and symptoms
(ASUI) score. Our study demonstrated a 0.25 improvement in
ASUI within 3–4 weeks, when weight loss was only 4%. In
studies using the ASUI a change of >0.25–0.3 is associated
with a clinically detectable difference in asthma severity
classification Although all these scoring systems could be
linked simply to weight loss the rapid change associated with
change in inflammatory markers is consistent with improve-
ment in asthma burden. The improvement in PEF of 44.6±
3.8 L/min in our study is consistent with the improvement
usually observed after “optimizing” controller medications in
mild/moderate asthmatics. Although major weight loss (13%)
is known to result in some improvement in pulmonary
function, our PEF improvement occurred within 3–4 weeks
when weight loss was 4%; PEF thereafter remained constant,
whereas continued through 8 weeks, suggesting that the change
Fig. 3. Alternate day calorie restriction results in changes in lipid and energy metabolism indicative of improved health in asthma subjects. Levels of total cholesterol
(a), 3-hydroxybutyrate (b), leptin (c), and ghrelin (d) were measured in serum samples from asthma subjects on successive ad libitum (AL) and CR days at baseline and
at 2, 4, and 8 weeks of ADCR. *P<0.05, **P<0.01 compared to the baseline value;##P<0.01 compared the corresponding CR value.
Results of analyses of nonpulmonary variables
Variable Baseline After 8 weeksChangeP value
670J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
in pulmonary function in response to the ADCR diet was not
due solely to weight loss.
The significant increase in the FEV1 after albuterol in the
subjects during the ADCR diet compared to baseline suggests
an effect of the ADCR diet on airway smooth muscle
responsiveness, consistent with an anti-inflammatory effect. In
a study of 58 obese women losing >13% of body weight over
6 months, there was no change in response to metacholine
challenge , suggesting that the changes were independent of
airway reactivity. Although we did not evaluate methacholine
responsiveness, the improved airway response to bronchodila-
tors should not be caused by weight loss per se and is consistent
with an anti-inflammatory response from the ADCR diet.
Particularly striking were the reductions in levels of TNFα,
BDNF, and markers of oxidative stress (protein carbonyls,
nitrotyrosine, and 8-isoprostane) in the serum of the asthma
patients during the course of the ADCR diet period. Levels of
these markers of inflammation and oxidative stress were
decreased on both ad libitum and CR days, indicating a
sustained effect of the ADCR diet that did not fluctuate in
response to the level of energy intake on the day prior to blood
sampling. The decreased levels of TNFα and BDNF suggest
that ADCR suppresses inflammation, which may contribute to
the beneficial effects of ADCR on asthma symptoms and
hyperresponsiveness. Indeed, studies of asthma patients and
animal models of asthma have provided evidence that TNFα
[10,12] and BDNF [16,39] are important mediators of airway
inflammation and associated symptoms. It was previously
reported that levels of protein carbonyls, nitrites and nitrates,
and lipid peroxidation products were increased in plasma from
patients with bronchial asthma compared to control subjects
. The consistent and progressive decrease in levels of
oxidative stress in our subjects may therefore be a marker of, or
to have contributed to, the improvement in symptoms on the
ADCR diet. The striking reduction in markers of oxidative
damage which we observed have not been described in daily
calorie restriction studies. Other authors have reported modest
or nonsignificant changes in levels of protein carbonyls with
various CR regimes [41–43]. Similarly, in a previous weight
loss study nitrotyrosine levels declined 23% in the Caucasian
women and remained unchanged in African American women
, suggesting that different groups of subjects exhibit
differential reductions in oxidative stress in response to weight
The mechanism(s) by which ADCR reduces oxidative stress
and inflammation in asthmatic subjects remains to be
established. However, based on previous studies of the effects
of alternate day fasting on cellular physiology in rodents, two
general mechanisms are likely. First, because subjects on
ADCR exhibit a reduction in overall energy intake and lose
weight, there is likely a reduction in cellular oxygen free radical
production [24,41,42]. The latter effect of ADCR would be
associated with lower levels of oxidatively modified proteins
and lipid peroxidation products in the blood. Second, ADCR
may impose a mild beneficial stress, to which cells respond
adaptively by up-regulating the expression of antioxidant
systems. Such increased cellular stress resistance has been
shown occur in rodents on an alternate day energy restriction
regimen, resulting in increased disease resistance . It will be
of considerable interest to determine the effects of ADCR on
gene expression in tissue involved in the pathogenesis of
We found that serum leptin levels were lower in subjects on
CR compared to AL days throughout the 8-week study period,
and that leptin levels on AL days decreased progressively
during the 8-week study period. Leptin has been shown to
exert proinflammatory actions , and it is therefore possible
that the reduction in leptin levels contributes to the anti-
Fig. 4. Markers of inflammation are reduced in asthma subjects in response to
the ADCR diet. Levels of TNF-α (a), BDNF (b), and ceramides (c) were
measured in serum samples from asthma subjects on successive ad libitum (AL)
and CR days at baseline and at 2, 4, and 8 weeks of ADCR. *P<0.05,
**P<0.01, ***P<0.001 compared to the baseline value.
671 J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
inflammatory effects of the ADCR diet. On the other hand, the
ADCR diet did not significantly affect circulating levels of
this hormone, a result consistent with our evidence that the
ADCR does not result in a sustained overactivation of the
Humans are unable to consistently comply with a long-term
daily caloric reduction of 40% (consuming 60% of main-
tenance), as has been used in most animals studies to date. The
authors of a recent 3-week trial in which 16 volunteers
alternated eating ad lib for 24 h and nothing the next 24 h
concluded that, due to persistent hunger and irritability, it was
unlikely subjects would stay on the regime for extended periods
of time . We designed the ADCR pattern of eating intended
as an accommodation to human needs and adaptation to human
meal pattern of the alternate day total fasting pattern used in
rodent studies. When rats or mice are maintained on an alternate
day fasting regimen they maintain body weights 10–25% lower
than ad libitum fed control animals, live up to 30% longer, and
exhibit improvement in a range of health indicators . A
regimen which allows ad libitum feeding on one day and
reduced food/caloric intake on the next day (for longer periods
of time), whereby a stable weight is maintained, may prolong
lifespan and healthspan in humans . Low levels of oxidative
stress may be necessary to reach very old age; at least two
studies have shown lower oxidative stress in centenarians than
in 70 year olds [49,50].
In our study, the ADCR pattern of eating consisted of
repeating cycles of a (approximately) 36-h period of very low
Fig. 5. Markers of oxidative stress are reduced in asthma subjects in response to the ADCR diet. Levels of total protein carbonyls (a), nitrotyrosine (b), 8-isoprostanes
(c), and lysine and histidine adducts of 4-hydroxynonenal (d) were measured in serum samples from asthma subjects on successive ad libitum (AL) and CR days at
baseline and at 2, 4, and 8 weeks of ADCR. *P<0.05, **P<0.01, ***P<0.001 compared to the baseline value.
Fig. 6. Levels of the antioxidant uric acid are increased in asthma subjects in
response to the ADCR diet. Levels of uric acid were measured in serum samples
from asthma subjects on successive ad libitum (AL) and CR days at baseline and
at 2, 4, and 8 weeks of ADCR. *P<0.05 compared to the baseline value.
672J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
calorie intake and a 12-h period of AL eating was tolerable and
efficacious in treating asthma symptoms, at least in obese
subjects. Larger studies that include a control group or a
crossover design with measures of airway reactivity and
inflammation will be required to further elucidate the full
impact of ADCR diets on obese asthma patients. Further studies
to improve asthma outcome are desirable since current therapies
do not seem to modify the underlying process or factors that
determine disease progression. It will also be important to
determine if such diets benefit patients with other disorders that
involve inflammation and oxidative stress such as athero-
sclerotic heart disease .
This research was supported, in part, by the National Institute
on Aging Intramural Research Program, NIH.
 Bel, E. H. Clinical phenotypes of asthma. Curr. Opin. Pulm. Med. 10:
 Ford, E. S. The epidemiology of obesity and asthma. J. Allergy Clin.
Immunol. 115:897–909; 2005.
 Shore, S. A.; Johnston, R. A. Obesity and asthma. Pharmacol. Ther. 110:
 Stenius-Aamiala, B.; Poussa, T.; Kvarnstrom, J.; Gronlund, E. L.; Yikahri,
M.; Mustajoki, P. Immediate and long term effects of weight reduction in
obese people with asthma: randomized controlled study. BMJ 320:
 Aaron, S. D.; Fergusson, D.; Dent, R.; Chen, Y.; Vandemheen, K. L.;
Dales, R. E. Effect of weight reduction on respiratory function and airway
reactivity in obese women. Chest 125:2046–2052; 2004.
 Schachter, L. M.; Salome, C. M.; Peat, J. K.; Woolcock, A. J. Obesity is a
risk for asthma and wheeze but not airway hyperresponsiveness. Thorax
 Andreadis, A. A.; Hazen, S. L.; Comhair, S. A.; Erzurum, S. C. Oxidative
and nitrosative events in asthma.Free Radic. Biol. Med. 35:213–225; 2003.
 Wood, L. G.; Gibson, P. G.; Garg, M. L. Biomarkers of lipid peroxidation,
airway inflammation and asthma. Eur. Respir. J. 21:177–186; 2003.
 Reynaert, N. L.; Ckless, K.; Wouters, E. F.; van der Vlie, A.; Janssen-
Heininger, Y. M. Nitric oxide and redox signaling in allergic airway
inflammation. Antioxid. Redox Signal. 7:129–143; 2005.
 Barnes, P. J. Cytokine-directed therapies for the treatment of chronic
airway diseases. Cytokine Growth Factor Rev. 14:511–522; 2003.
 Shakoory, B.; Fitzgerald, S. M.; Lee, S. A.; Chi, D. S.; Krishnaswamy, G.
The role of human mast cell-derived cytokines in eosinophil biology.
J. Interferon Cytokine Res. 24:271–281; 2004.
 Halasz, A.; Cserhati, E.; Magyar, R.; Kovacs, M.; Cseh, K. Role of TNF-
alpha and its 55 and 75 kDa receptors in bronchial hyperreactivity. Respir.
Med. 96:262–267; 2002.
 Yokoyama, A.;Kohno,N.; Fujino,S., et al. Circulating interleukin-6 levels
in patients with bronchial asthma. Am. J. Respir. Crit. Care Med. 151:
 Sharma, A.; Bansal, S.; Nagpal, R. K. Lipid peroxidation in bronchial
asthma. Indian J. Pediatr. 70:715–717; 2003.
 Virchow, J. C.; Julius, P.; Lommatzsch, M.; Luttmann, W.; Renz, H.;
Braun, A. Neurotrophins are increased in bronchoalveolar lavage fluid
after segmental allergen provocation. Am. J. Respir. Crit. Care Med. 158:
 Noga, O.; Hanf, G.; Schaper, C.; O'Connor, A.; Kunkel, G. The influence
of inhalative corticosteroids on circulating nerve growth factor, brain-
derived neurotrophic factor and neurotrophin-3 in allergic asthmatics.
Clin. Exp. Allergy 31:1906–1912; 2001.
 Hvizdos, K. M.; Jarvis, B. Budesonide inhalation suspension: a review of
its use in infants, children and adults with inflammatory respiratory
disorders. Drugs 60:1141–1178; 2000.
 Rossi, G. A.; Cerasoli, F.; Cazzola, M. Safety of inhaled corticosteroids:
room for improvement. Pulm. Pharmacol. Ther. [Epub ahead of print];
2005 (Dec 13).
 Heilbronn, L. K.; Ravussin, E. Calorie restriction and aging: review of the
literature and implications for studies in humans. Am. J. Clin. Nutr. 78:
 Mattison, J. A.; Lane, M. A.; Roth, G. S.; Ingram, D. K. Calorie restriction
in rhesus monkeys. Exp. Gerontol. 38:35–46; 2003.
 Fontana, L.; Meyer, T. E.; Klein, S.; Holloszy, J. O. Long-term calorie
restriction is highly effective in reducing the risk for atherosclerosis in
humans. Proc. Natl. Acad. Sci. U. S. A. 101:6659–6663; 2004.
 Goodrick, C. L.; Ingram, D. K.; Reynols, M. A.; Freeman, J. R.; Cider,
N. L. Effects of intermittent feeding upon growth and life span in rats.
Gerontology 28:233–241; 1982.
 Anson, R. M.; Guo, Z.; de Cabo, R., et al. Intermittent fasting dissociates
beneficial effects of dietary restrictionon glucose metabolismandneuronal
resistance to injury from calorie intake. Proc. Natl. Acad. Sci. U. S. A. 100:
 Mattson, M. P.; Wan, R. Beneficial effects of intermittent fasting and
caloric restriction on the cardiovascular and cerebrovascular systems.
J. Nutr. Biochem. 16:129–137; 2005.
 Mattson, M. P. The need for controlled studies of the effects of meal
frequency on health. Lancet 365:1978–1980; 2005.
 Scheen, A. J. Results of obesity treatment. Ann. Endocrinol. (Paris) 63:
 National Asthma Education and Prevention Program (NAEPP). Expert
Panel Report 2: Guidelines for the Diagnosis and Management of Asthma.
National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD. publication No. 97-4051; 1997.
 Revicki, D. A.; Leidy, N. K.; Brennan-Diemer, F.; Sorensen, S.; Togias, A.
Integrating patient preferences into health outcomes assessment: the
multiattribute Asthma Symptom Utility Index. Chest 114:998–1007;
 Lee, M.; Hyun, D.; Jenner, P.; Halliwell, B. Effect of overexpression of
wild-type and mutant Cu/Zn-superoxide dismutases on oxidative damage
and antioxidant defences: relevance to Down's syndrome and familial
amyotrophic lateral sclerosis. J. Neurochem. 76:957–965; 2001.
 Hyun, D. H.; Gray, D. A.; Halliwell, B.; Jenner, P. Interference with
ubiquitination causes oxidative damage and increased protein nitration:
implications for neurodegenerative diseases. J. Neurochem. 90:422–430;
 Cutler, R. G.; Kelly, J.; Storie, K., et al. Involvement of oxidative stress-
induced abnormalities in ceramide and cholesterol metabolism in brain
aging and Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A.
 Knopp, R. H.; Magee, M. S.; Raisys, V.; Benedetti, T.; Bonet, B.
Hypocaloric diets and ketogenesis in the management of obese gestational
diabetic women. J. Am. Coll. Nutr. 10:649–667; 1991.
 Sharma, V.; McNeill, J. H. The emerging roles of leptin and ghrelin in
cardiovascular physiology and pathophysiology. Curr. Vasc. Pharmacol.
 Haughey, N. J.; Cutler, R. G.; Tamara, A., et al. Perturbation of
sphingolipid metabolism and ceramide production in HIV-dementia.
Ann. Neurol. 55:257–267; 2004.
 Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Sphingolipids in inflam-
mation: roles and implications. Curr. Mol. Med. 4:405–418; 2004.
 Summers, S. A. Ceramides in insulin resistance and lipotoxicity. Prog.
Lipid Res. 45:42–72; 2006.
 Glantzounis, G. K.; Tsimoyiannis, E. C.; Kappas, A. M.; Galaris, D. A.
Uric acid and oxidative stress. Curr. Pharm. Des. 11:4145–4151; 2005.
 O'Connor, R. D.; Gilmore, A.S.; Manjunath, R.; Stanford, R. H.;Legorreta,
A. P.; Jhingran, P. M. Comparing outcomes in patients with persistent
asthma: a registry of two therapeutic alternatives. Curr. Med. Res. Opin.
 Lommatzsch, M.; Schloetcke, K.; Klotz, J., et al. Brain-derived neuro-
673J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674
trophic factor in platelets and airflow limitation in asthma. Am. J. Respir. Download full-text
Crit. Care Med. 171:115–120; 2005.
 Nadeem, A.; Chhabra, S. K.; Masood, A.; Raj, H. G. Increased oxidative
stress and altered levels of antioxidants in asthma. J. Allergy Clin.
Immunol. 111:72–78; 2003.
 Heilbronn,L. K.; de Jong, L.; Frisard, M. I., et al. Effect of 6-month calorie
restriction on biomarkers of longevity, metabolic adaptation, and oxidative
stress in overweight individuals: a randomized controlled trial. JAMA
 Dandona, P.; Mohanty, P.; Ghanim, H., et al. The suppressive effect of
dietary restrictionandweight loss in the obese on the generationof reactive
oxygen species by leukocytes, lipid peroxidation, and protein carbonyla-
tion. J. Clin. Endocrinol. Metab. 86:355–362; 2001.
 Zainal, T. A.; Oberley, T. D.; Allison, D. B.; Szweda, L. I.; Weindruch, R.
Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal
muscle. FASEB J. 14:1825–1836; 2000.
 Fenster, C. P.; Darley-Usmar, V. M.; Landar, A. L., et al. Weight loss and
race modulate nitric oxide metabolism in overweight women. Free Radic.
Biol. Med. 37:695–702; 2004.
 Matarese, G.; Moshchos, S.; Mantzoros, C. S. Leptin in immunology.
J. Immunol. 174:3137–3142; 2005.
 Heilbronn, L. K.; Civitarese, A. E.; Bogacka, I.; Smith, S. R.; Hulver, M.;
Ravussin, E. Glucose tolerance and skeletal muscle gene expression in
response to alternate day fasting. Obes. Res. 13:574–581; 2005.
 Mattson, M. P. Energy intake, meal frequency, and health: a neurobiolo-
gical perspective. Annu. Rev. Nutr. 25:237–260; 2005.
 Johnson, J. B.; Laub, D. R.; John, S. The effect on health of alternate day
calorie restriction: eating less and more than needed on alternate days
prolongs life. Med. Hypotheses 67 (2):209–211; 2006.
 Suzuki, M.; Wilcox, B. J.; Wilcox, C. D. Implications from and for food
cultures for cardiovascular disease: longevity. Asia Pac. J. Clin. Nutr 10:
 Paolisso, G.; Tagliamonte, M. R.; Rizzo, M. R.; Manzella, D.;
Gambardella, A.; Varricchio, M. Oxidative stress and advancing age:
results in healthy centenarians. J. Am. Geriatr. Soc. 46:833–838; 1998.
 Shishehbor, M. H.; Aviles, R. J.; Brennan, M. L., et al. Association of
nitrotyrosine levels with cardiovascular disease and modulation by statin
therapy. JAMA 289:1675–1680; 2003.
674J.B. Johnson et al. / Free Radical Biology & Medicine 42 (2007) 665–674