Report

A Diet Mimicking Fasting Promotes Regeneration

and Reduces Autoimmunity and Multiple Sclerosis
Symptoms
Graphical Abstract Authors
In Young Choi, Laura Piccio,
Patra Childress, ..., Friedemann Paul,
Markus Bock, Valter D. Longo

Correspondence
vlongo@usc.edu

In Brief
Choi et al. show that cycles of a fasting
mimicking diet (FMD) ameliorate disease
severity by suppressing autoimmunity
and stimulating remyelination via
oligodendrocyte regeneration in multiple
sclerosis (MS) mouse models. They also
show that a similar FMD is a safe, feasible,
and possibly a potentially effective
treatment for patients with relapsing-
remitting MS.

Highlights
d FMD reduces pro-inflammatory cytokines and increases
corticosterone levels

d FMD suppresses autoimmunity by inducing lymphocyte

apoptosis

d FMD promotes regeneration of oligodendrocyte in multiple

MS models

d FMD is a safe, feasible, and potentially effective treatment for

MS patients

Choi et al., 2016, Cell Reports 15, 2136–2146

June 7, 2016 ª 2016 The Author(s).
http://dx.doi.org/10.1016/j.celrep.2016.05.009
 Cell Reports

Report

A Diet Mimicking Fasting Promotes

Regeneration and Reduces Autoimmunity
and Multiple Sclerosis Symptoms
In Young Choi,1,10 Laura Piccio,2,10 Patra Childress,3 Bryan Bollman,2 Arko Ghosh,4 Sebastian Brandhorst,1
Jorge Suarez,1 Andreas Michalsen,5 Anne H. Cross,2 Todd E. Morgan,1 Min Wei,1 Friedemann Paul,6,7 Markus Bock,6,7,11
and Valter D. Longo1,4,8,9,11,*
1Longevity Institute, School of Gerontology, and Department of Biological Sciences, University of Southern California, Los Angeles,

CA 90089, USA
2Department of Neurology and Neurosurgery and Hope Center for Neurological Disorders, Washington University School of Medicine,

St. Louis, MO 63110, USA

3Global Medicine Program, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
4Department of Neuroscience, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles,

CA 90089, USA
5Institute of Social Medicine, Epidemiology and Health Economics, Charité University Medicine Berlin, 10117 Berlin, Germany
6NeuroCure Clinical Research Center and Clinical and Experimental Multiple Sclerosis Research Center, Department of Neurology, Charité

University Medicine Berlin, 10117 Berlin, Germany

7Experimental and Clinical Research Center, a joint cooperation between the Charité Medical Faculty and the Max-Delbrueck Center for

Molecular Medicine, 10117 Berlin, Germany

8Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern

California, Los Angeles, CA 90089, USA

9IFOM, FIRC Institute of Molecular Oncology, 20139 Milan, Italy
10Co-first author
11Co-senior author

*Correspondence: vlongo@usc.edu
http://dx.doi.org/10.1016/j.celrep.2016.05.009

SUMMARY INTRODUCTION

Dietary interventions have not been effective in the
treatment of multiple sclerosis (MS). Here, we show
that periodic 3-day cycles of a fasting mimicking
diet (FMD) are effective in ameliorating demyelin-
ation and symptoms in a murine experimental auto-
immune encephalomyelitis (EAE) model. The FMD
reduced clinical severity in all mice and completely
reversed symptoms in 20% of animals. These
improvements were associated with increased
corticosterone levels and regulatory T (Treg) cell
numbers and reduced levels of pro-inflammatory
cytokines, TH1 and TH17 cells, and antigen-present-
ing cells (APCs). Moreover, the FMD promoted
oligodendrocyte precursor cell regeneration and
remyelination in axons in both EAE and cuprizone
MS models, supporting its effects on both sup-
pression of autoimmunity and remyelination. We
also report preliminary data suggesting that an
FMD or a chronic ketogenic diet are safe, feasible,
and potentially effective in the treatment of re-
lapsing-remitting multiple sclerosis (RRMS) patients
(NCT01538355).
Multiple sclerosis (MS) is an autoimmune disorder characterized
by T cell-mediated demyelination and neurodegeneration in the
CNS (Friese and Fugger, 2005; Pender and Greer, 2007; Sospedra
and Martin, 2005). In experimental autoimmune encephalomyelitis
(EAE), an animal model for MS, activated myelin-specific TH1 and
TH17 cells cross the blood-brain barrier and migrate into the CNS,
where they are activated by local antigen-presenting cells (APCs)
and promote inflammation (Dhib-Jalbut, 2007; Fletcher et al.,
2010; Goverman, 2009; Hemmer et al., 2002). This inflammatory
process leads to oligodendrocyte death, demyelination, and
axonal damage, which eventually cause neurologic damage (Luc-
chinetti et al., 1999; Raine and Wu, 1993). Although oligodendro-
cyte precursor cells (OPCs) can migrate to the sites of MS lesions,
they often fail to differentiate into functional oligodendrocytes
(Chang et al., 2002; Wolswijk, 1998). Several MS treatment drugs
have been effective in reducing immune responses, but their
impact on long-term disease progression, accrual of irreversible
neurological disability, and immune system function remains
largely unclear, underlining the need for novel therapeutic strate-
gies (Wingerchuk and Carter, 2014). Therefore, effective treat-
ments for MS may require not only the mitigation of autoimmunity
but also the stimulation of oligodendrocyte regeneration and resto-
ration of a functional myelin sheath. Periodic cycles of prolonged
fasting (PF) or of a fasting mimicking diet (FMD) lasting 2 or more

2136 Cell Reports 15, 2136–2146, June 7, 2016 ª 2016 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Figure 1. FMD Cycles Decrease Disease Severity of the MOG35–55-Induced EAE Model
(A) Diagram displaying the time course of the immunization and the diet interventions.
(B) The EAE severity scores of the control diet (EAE CTRL; n = 23), ketogenic diet (EAE KD; n = 13), semi-therapeutic FMD cycles (EAE FMD (S); n = 7), or
therapeutic FMD cycles (FMD(T); n = 23).
(C) Incidence rate of EAE CTRL and EAE FMD (S) (n = 7–23).
(D) EAE severity score in mice for which FMD(T) completely reversed EAE severity, with no observable disease (score = 0; 5 out of 23 mice).
(E) EAE severity score of the best-performing control mice (n = 12) and FMD(T) mice (n = 12).
(F) EAE severity score of the mice treated with FMD after chronic EAE development (EAE CTRL-FMD; n = 6).
(G–M) Spinal cord sections of EAE CTRL and EAE FMD (T) mice with quantification of H&E staining (G), solochrome cyanine staining (H), and MBP (myelin basic
protein)/SMI32 (I) double staining of spinal cord sections isolated at day 14.
Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test, one-way or two-way ANOVA, and Bonferroni post test. Scale bar
represents 200 mm.

days can increase protection of multiple systems against a variety
of chemotherapy drugs in mice and possibly humans (Fontana
et al., 2010; Guevera-Aguirre et al., 2011; Lee et al., 2010; Longo
and Mattson, 2014). Moreover, PF or an FMD reverses the immu-
nosuppression or immunosenescence of either chemotherapy
or aging through hematopoietic stem cell-based regeneration
(Brandhorst et al., 2015; Cheng et al., 2014). Chronic caloric re-
striction, a ketogenic diet (KD), and intermittent fasting have
been shown to help prevent EAE by reducing inflammation and
enhancing neuroprotection when administered prior to disease
induction or signs (Esquifino et al., 2007; Kafami et al., 2010; Kim
do et al., 2012; Piccio et al., 2008), but dietary interventions have
not been reported to be effective as therapies for EAE or MS or
to promote myelin regeneration.
Here, we report on the effects of low-calorie and low-protein
FMD cycles as a treatment in MS mouse models, and we inves-
tigate the mechanisms involved. Furthermore, we report prelim-
inary results on the safety and feasibility of a FMD and a KD in
patients with relapsing-remitting multiple sclerosis (RRMS).
RESULTS
FMD Cycles Reduce Disease Severity in the MOG35–55-
Induced EAE Model
We examined the effects of 3 cycles of a very-low-calorie and
low-protein FMD lasting 3 days every 7 days or a KD continued
for 30 days in EAE-induced by active immunization with myelin
oligodendrocyte glycoprotein 35–55 (MOG35–55) (Figure 1A).

Cell Reports 15, 2136–2146, June 7, 2016 2137

Groups of mice were treated semi-therapeutically (EAE FMD (S),
where FMD treatment started after 10% of the immunized pop-
ulation showed signs of EAE) or therapeutically (EAE FMD (T),
where FMD treatment started after all of the immunized popula-
tion showed signs of EAE). FMD and KD treatment decreased
disease severity compared to that in the control group (Fig-
ure 1B). However, the FMD reduced the mean severity score
to 1, whereas a KD reduced the severity score to 2 at the later
stages (Figure 1B). In the EAE FMD (S) group, FMD treatment not
only delayed the onset of disease but also lowered the incidence
rate (100% versus 45.6%; Figure 1C). In the EAE FMD (T) group,
FMD cycles completely reversed the severity score to 0 in 21.7%
of the cohort (no observable signs; Figure 1D) and reduced the
severity score to <0.5 in >50% of mice (12 out of 23 mice; Fig-
ure 1E). To address whether the FMD cycles also have beneficial
effects in chronic EAE models that have established disease, we
initiated FMD treatment 2 weeks after the initial signs of EAE
were observed (EAE CTRL-FMD). Prior to treatment, the control
diet EAE group (EAE CTRL) and EAE CTRL-FMD cohorts had
similar severity scores (3.19 ± 0.52 versus 3.30 ± 0.27; day 24).
After three FMD cycles, we observed a significant reduction in
severity score in the EAE CTRL-FMD cohort compared to the
EAE CTRL cohort (3.3 ± 0.57 versus 2.1 ± 0.89; day 42; p <
0.05; Figure 1F). As infiltration of immune cells and demyelination
are histopathological hallmarks of EAE and MS, spinal cord sec-
tions from control and FMD(T) mice were stained with H&E to
visualize infiltrating immune cells (Figure 1G) or solochrome
cyanine to visualize myelin (Figure 1H). To assess demyelination
and axonal damage, immunohistochemistry was performed us-
ing antibodies against myelin basic protein (MBP) or dephos-
phorylated neurofilaments (SMI-32; Figure 1I). At day 3, levels
of infiltrating immune cells and demyelination were similar in
the EAE CTRL and EAE FMD groups (Figures 1J and S1H). At
day 14, sections of EAE CTRL mice displayed severe immune
cell infiltration corresponding to demyelinated lesions, reduced
MBP expression, and increased SMI-32 expression (Figures
1J–1M). By contrast, sections of EAE FMD mice at day 14 dis-
played significantly reduced immune cell infiltration and demye-
lination (Figures 1J–1M). Although MBP staining showed no sig-
nificant difference between EAE CTRL and EAE FMD mice at day
14 (Figure 1L), neurofilament dephosphorylation in EAE FMD
mice was reduced compared to EAE CTRL mice (Figure 1M).
Overall, these results suggest that FMD cycles reduce EAE dis-
ease severity in part by reducing inflammation and preventing
demyelination and axonal damage.
FMD Cycles Reduce Infiltration of Immune Cells in the
Spinal Cord
To investigate the capacity of FMD cycles to reduce potential
autoimmune T cells, we measured circulating white blood cells
(WBCs), lymphocytes, monocytes, and granulocytes in naive,
EAE CTRL, EAE FMD, and EAE FMD:RF (measured 4 days after
returning to a standard ad lib diet) mice after three cycles of the
FMD regimen (Figure 2A). The FMD resulted in a temporary
40%–50% reduction in total WBCs, lymphocytes, monocytes,
and granulocytes. Upon returning to the standard ad lib diet
(EAE FMD:RF), all complete blood counts (CBCs) returned to
either naive or lower levels than those observed in the EAE
2138 Cell Reports 15, 2136–2146, June 7, 2016
CTRL, with the exception of granulocytes, indicating that the
FMD cycles cause both WBC death and regeneration (Figure 2A).
Next, we measured the inflammatory markers associated with
EAE pathophysiology. Day 3 and day 14 spinal cord sections
of the EAE CTRL mice were extensively populated with
CD11b+ cells (Figure 2B). However, at day 14, the EAE FMD
mice displayed a 75% reduction (p < 0.05) in spinal cord-associ-
ated CD11b+ cells compared to mice on the control diet (11.7%
versus 2.8%; Figure 2B). Since myelin-specific effector T cells
migrate into the CNS and initiate demyelination, we investigated
the accumulation of CD4+ or CD8+ T cells in the spinal cord.
A large number of CD4+ T cells were detected in the white matter
of spinal cord sections from the control diet cohort (Figure 2C). In
contrast, the FMD-treated cohort displayed a >4-fold reduction
(p < 0.01) in CD4+ T cells at day 3 (8.6% versus 1.5%; Figure 2C)
compared to the control diet cohort, which remained lower even
at day 14. The FMD group also had reduced CD8+ T cells (day 3:
1.3% versus 0.4%; p < 0.01; Figure 2D) compared to the control
diet group. To investigate whether the FMD affects APCs, we
isolated splenocytes from EAE CTRL and EAE FMD mice at
day 3, stained them for CD11c and F4/80, and characterized
them by flow cytometry. We observed a significant decrease
(p < 0.05) in CD11c+ dendritic cells in the EAE FMD cohort
compared to the EAE CTRL cohort (3.08% ± 0.70% versus
1.46% ± 0.31%), but we did not observe any changes in the
number of F4/80+ macrophage cells in the control or FMD-
treated groups (Figures 2E and S2B). To determine the effects
of the FMD treatment on T cell infiltration in the spinal cord, we
measured T cell activation levels. The number of CD4+ T cells
and CD8+ T cells in EAE CTRL and EAE FMD mice was similar
(Figures S2C and S2D), but the ratio of splenic naive (CD44low)
to activated (CD44high) CD4+ T cells was increased (p < 0.05) in
the FMD group compared to the control group (1.95 versus
3.67; Figure 2F). No difference in CD8+ T cells was observed (Fig-
ure S2E). Moreover, the total number of effector (CD44high and
CD62Llow) T cells was reduced in the FMD compared to the con-
trol group, but the ratio of effector (CD44high and CD62Llow) to
memory T (CD44high and CD62Lhigh) cells did not change (Fig-
ures S2F–S2H). These results indicate that FMD cycles reduce
the number of dendritic cells and increase the relative number
of naive T cells, which may explain the reduced autoimmunity
caused by the FMD.
FMD Cycles Induce Autoreactive Lymphocyte Apoptosis
and Increase the Number of Naive Cells
To determine whether FMD cycles also reduce the number of
MOG-specific antigen-reactive cells, we used a major histocom-
patibility complex (MHC) tetramer (MOG35–55/IAb) to identify
antigen-reactive cells after an FMD cycle in vivo. The number
of CD4+ MOG35–55 /IAb+ cells was reduced in the EAE FMD
cohort compared to the EAE CTRL cohort (5.75% ± 0.51%
versus 3.83% ± 0.66% of lymphocytes; * p < 0.05; Figure 2G).
To determine whether the reduced active T cell number is
due to an increase in the number of regulatory T (Treg) cells, we
isolated lymphocytes from draining lymph nodes and spleens
of EAE CTRL or EAE FMD mice and analyzed them for CD4+
CD25+ FoxP3+ Treg cells. The FMD cohort showed a 2-fold in-
crease (p < 0.01) in the number of CD25+ FoxP3+-expressing
 (legend on next page)

Cell Reports 15, 2136–2146, June 7, 2016 2139

Treg cells (13.6% ± 4.2% versus 25.1% ± 4.2%; Figure 2H).
Moreover, the FMD cohort showed a 27.8% reduction (p <
0.05) in the number of interferon g (IFN-g)- expressing TH1 cells
(2,974.4 ± 708.0 versus 2,148.1 ± 1,396.1; Figure 2I) and a
46.5% reduction (p < 0.05) in the number of interleukin-17
(IL-17)-expressing TH17 cells (2,535.9 ± 722.0 versus 1,357.1 ±
256.2; Figure 2J), both of which are known to be central media-
tors of EAE. Interestingly, upon re-feeding of the control diet, the
EAE FMD treatment group (EAE FMD:RF) showed a 72.9%
reduction (p < 0.05) in the number of IFN-g-expressing TH1 cells
(2,974.4 ± 708.0 versus 805.8 ± 251.5; Figure 2I) and a 82.9%
reduction (p < 0.05) in the number of IL-17-expressing TH17 cells
(2,535.9 ± 722.0 versus 432.4 ± 117.4; Figure 2J), suggesting
that the FMD can prevent autoimmunity in part by reducing the
levels of pro-inflammatory T cells implicated in EAE.
In order to assess how FMD cycles may reduce the number of
T cells, we measured apoptosis in MOG-specific T cells (CD3+
MOG35–55/IAb) in vivo. We observed a significant increase (p <
0.05) in apoptotic CD3+ MOG35–55/IAb levels in the EAE FMD
cohort compared to the EAE CTRL cohort (28.3% ± 4.94%
versus 39.1% ± 4.79%; Figure 2K), which was consistent with
the major reduction in the number of WBCs and lymphocytes
observed in the FMD group (Figure 2A). To investigate whether
these apoptotic cells are replaced by newly generated cells,
we treated the mice with bromodeoxyuridine (BrdU) during the
re-feeding period (four injections within 48 hr, at 1 mg of BrdU
per injection). Splenocytes were isolated 4 days after the re-
feeding of the regular diet and stained for BrdU (Figure S2I).
We observed no difference in levels of total BrdU+ lymphocytes
(8.11% ± 1.99% versus 12.02% ± 2.72%; Figure S2J), but we
observed a significantly reduced proliferation of TH1 (BrdU+
CD4+IFNg+) (5.74% ± 1.07% versus 3.65% ± 0.63%; *p <
0.05; Figure 2L) and no difference in proliferation of TH17
(BrdU+CD4+IL17+) (4.71% ± 1.53% versus 5.01% ± 1.66%; Fig-
ure S2K). Taken together, these data indicate that FMD cycles
may promote apoptosis of autoreactive T cells, leading to an
increase in the proportion of naive T cells and regulatory
T cells. In addition, FMD cycles may interfere with proliferation
and differentiation of TH1 cells, but not TH17 cells. To investigate
whether the FMD’s effects on CNS infiltrating immune cells are
associated with suppression of TH1- and TH17-dependent cyto-
kine production (IL-17, IFN-g, and tumor necrosis factor a
[TNF-a]), we analyzed serum from naive, EAE CTRL, and EAE
FMD mice (Figures 2K–2M). We observed significant reductions
in serum TNF-a (113.3 ± 7.9 versus 79.3 ± 10.5 pg/ml; p < 0.001;
Figure 2M), IFN-g (558.43 ± 124.5 versus 296.0 ± 83.4 pg/ml;
p < 0.001; Figure 2N), and IL-17 (36.8 ± 9.67 versus 20.75 ±
4.2 pg/ml; p < 0.01; Figure 2O). To identify a potential mediator
for the effects of FMD cycles on the suppression of autoimmune
responses, we measured serum corticosterone levels. Cortico-
sterone is a glucocorticoid hormone with broad anti-inflamma-
tory and immunosuppressive effects affecting leukocyte dis-
tribution, trafficking, and death (Ashwell et al., 2000; Herold
et al., 2006; Planey and Litwack, 2000; Vegiopoulos and Herzig,
2007). Serum corticosterone levels were elevated in association
with the first signs of EAE (EAE day 1, before treatment) (data not
shown). FMD treatment caused a further increase in corticoste-
rone levels at day 3 compared to those of controls (245.9 ± 38.8
versus 375.0 ± 94.1 ng/ml; p < 0.01), which returned to EAE basal
levels by day 14 in both groups (Figure 2P). These results indi-
cate that FMD cycles reduce the number of TH1 and TH17
effector cells and the production of pro-inflammatory cytokines.
These effects of the FMD may be regulated in part by the tempo-
rary elevation of corticosterone levels, dampening of T cell acti-
vation, and reduced APC and T cell infiltration in the spinal cord.
FMD Reverses EAE Symptoms by Reducing the Level
and Reactivity of Established Autoimmune Cells
To determine how the FMD affects the initiation of EAE, spleno-
cytes were isolated from EAE CTRL and EAE FMD mice, re-acti-
vated with MOG35–55 peptide and IL-23 ex vivo, and transferred
into naive recipient mice to induce EAE. The mice were then sub-
jected to either a control diet or FMD cycles (Figure 3A). The
supernatant from ex vivo splenocyte cultures derived from
EAE FMD mice showed no difference in TNF-a levels (110.8 ±
14.9 pg/ml versus 97.1 ± 8.4 pg/ml; Figure 3B) but a major reduc-
tion (p < 0.01) in the levels of IFN-g (342.0 ± 29.8 pg/ml versus 46.6
± 16.6 pg/ml Figure 3C) and IL-17 (850.5 ± 442.0 pg/ml versus
257.4 ± 36.4 pg/ml; Figure 3D). Interestingly, upon in vitro reactiva-
tion, both EAE CTRL and EAE FMD had similar levels of TH1 and
TH17 differentiated cells (Figures 3E and 3F). To determine
whether the immune cells from EAE CTRL and EAE FMD mice

Figure 2. FMD Cycles Decrease the Number of Infiltrating T Cells in the Spinal Cord
(A) Total white blood cell (WBC), lymphocyte, monocyte, and granulocyte counts of naive, EAE-CTRL, EAE-FMD, and EAE-FMD:RF (after 3 days of re-feeding)
mice after three cycles of the FMD and a matched time point for EAE-CTRL mice.
(B–D) Spinal cord sections (day 14) and quantification at days 3 and 14 after the first sign of EAE for CD11b+ (B), CD4+ (C), and CD8+ (D) (at least six sections per
mouse).
(E) CD11c+ isolated from EAE CTRL or EAE FMD mice on day 3, and quantification of cells from the total isolated splenocyte.
(F) CD4+ gated for CD44low or CD44high cells isolated from EAE CTRL or EAE FMD mice, and quantification of percent splenocytes in CD4+ CD44low (inactive) or
CD4+ CD44high (active) cells.
(G) CD3+ lymphocytes gated for CD4 and MOG35–55/IAb from EAE CTRL or EAE FMD mice, and quantification of MOG-specific CD4+ cells.
(H) CD4+ CD25+ FoxP3+ isolated from EAE CTRL or EAE FMD mice, and quantification of CD25+ FoxP3+ in CD4+ cells.
(I and J) Intracellular staining for either IFNg (I) or IL17(J) after gated for CD4+ of the naive, EAE CTRL, EAE FMD, EAE FMD:RF and quantification of cell counts.
(K) Quantification of Annexin V+ apoptotic CD3+ MOG35-55/IAb cells.
(L) Quantification of CD4+IFNg+ of BrdU+ lymphocytes.
(M–O) Serum TNF-a (M), IFN-g (N), and IL-17 levels (O) (pg/ml) in naive, EAE CTRL, and EAE FMD mice on day 3 after the first sign of EAE.
(P) Serum corticosterone levels (ng/ml) before immunization, at the time of symptom occurrence, or 3 or 14 days after the initial symptom appeared in the control
or FMD group.
n = 4–8 per group; mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, Student’s t test, one-way ANOVA, and Bonferroni post test. Scale bar represents 200 mm.

2140 Cell Reports 15, 2136–2146, June 7, 2016


have similar encephalitogenic effects, we transferred splenocytes
from either donor group (EAE CTRL or EAE FMD) into naive recip-
ients (A, EAE CTRL donor to control diet recipient; and C, EAE FMD
donor to control diet recipient). This resulted in a similar disease
incidence rate (Figure 3G) and an equally severe EAE disease
severity by day 20 (2.38 ± 0.48 versus 2.70 ± 0.75; Figure 3H), indi-
cating that the FMD did not affect the development and function of
reactive immune cells in vivo or ex vivo. However, when FMD treat-
ment was initiated after transfer of control donor splenocytes (B,
EAE CTRL donor to naive mice with FMD treatment), recipient
mice displayed a delayed disease onset (day 12 versus day 16
Figure 3. Antigen-Activated Splenocytes
from EAE-CTRL and the EAE-FMD Mice
Had Similar Encephalitogenic Effects
(A) Diagram for the adoptive transfer EAE model.
(B–D) Quantification of TNF-a (B), IFN-g (C), and
IL-17 (D) (pg/ml) in the supernatant from ex vivo
cultures of splenocytes from naive, EAE CTRL,
and EAE FMD mice either with or without MOG35–55
and IL-23 re-activation.
(E and F) Quantification of TH1 or TH17 (repre-
sented by percentage of CD4+) from lymphocyte
culture of EAE CTRL and EAE FMD mice with or
without MOG35–55 and IL-23 re-activation.
(G) Incidence rate of adoptive transfer EAE
groups.
(H) EAE severity score of adoptive transfer EAE
groups.
n = 5–6 per group; mean ± SEM. *p < 0.05, **p <
0.01, and ***p < 0.001, Student’s t test, one-way
ANOVA, and Bonferroni post test.
post-transfer; Figure 3G) and a major
reduction in EAE severity scores
compared to control mice (2.38 ± 0.48
versus 0.75 ± 0.87; Figure 3H). Taken
together, these results suggest that T cell
priming in response to myelin antigen
occurred normally in the EAE CTRL and
EAE FMD groups, but the FMD can reduce
the level of the existing autoimmunity.
FMD Cycles Stimulate
Remyelination by Promoting
Oligodendrocyte Regeneration
To investigate whether the reduced demy-
elination in FMD mice may also be related
to enhanced oligodendrocyte regenera-
tion, we first carried out a quantitative im-
age analysis of NG2+ (an oligodendrocyte
progenitor cell [OPC] marker) and GST-
p+ (a mature oligodendrocyte marker) in
spinal cord sections from control or FMD
mice (Figure 4A). We observed no differ-
ence in the number of NG2+ OPCs in sec-
tions taken from EAE CTRL and EAE FMD
mice (Figure S3A). However, at day 14, the
number of GST-p+ oligodendrocytes was
reduced in the EAE CTRL group, but not in the EAE FMD group
(886.7 ± 41.6 versus 1,273 ± 200.3; cells per spinal cord section
area; p < 0.01; Figure 4B). To assess whether the normal levels
of mature oligodendrocytes in the EAE FMD group were due to
enhanced regeneration and/or differentiation, EAE CTRL or EAE
FMD mice were injected with BrdU at the time of re-feeding
(day 10). We observed a major increase (p < 0.01) in the percent-
age of cells that are double positive for BrdU+ and GST-p+ in the
EAE FMD group compared to the EAE CTRL group (42.9% ±
11.2% versus 83.0% ± 13.2%; p < 0.01), suggesting that the
FMD promotes oligodendrocyte differentiation from precursor
Cell Reports 15, 2136–2146, June 7, 2016 2141
Figure 4. FMD, which Protects the Mouse Spinal Cord from Loss of Oligodendrocytes and Enhances Remyelination, Is Safe and Potentially
Effective in the Treatment of MS Patients
(A–C) Spinal cord sections isolated at day 14 and quantification for GST-p (mature oligodendrocyte) and BrdU (A), TUNEL and NG2 (oligodendrocyte precursor
cells) (B), and TUNEL and GST-p (C) in naive, EAE-CTRL, or EAE-FMD mice.
(F–H) Sections from the corpus callosum region and quantification of cuprizone treated brains, stained with Luxol Fast Blue of the naive control, end of 5 weeks of
cuprizone diet (week 0), cuprizone (5 weeks) plus regular chow (2 weeks), and cuprizone (5 weeks) plus FMD cycle (2 weeks).
(I and J) Section from the corpus callosum region and its quantification of the cuprizone treated brains stained with GST-p+ of cuprizone (5 weeks) plus regular
chow (2 weeks), and cuprizone (5 weeks) plus FMD (2 weeks). Quantification is normalized to percent naive GST-p+ level.
(K–N) Change in quality of life at 3 months in terms of overall quality of life (K), change in health (L), physical health composite (M), and mental health composite (N).
The dotted line represents a threshold that is thought to be clinically important (R5 points). Data represent mean ± standard error of the difference (SED);
*p < 0.05, Mann-Whitney U test. An increase of R5 points is considered clinically important.
At least 12 sections per mouse were used for quantification; n = 4; mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA and Bonferroni post test.

2142 Cell Reports 15, 2136–2146, June 7, 2016

cells (Figure 4C). To assess the effects of the FMD on either OPCs
or mature oligodendrocytes, sections were stained with TUNEL,
an apoptotic marker, and GST-p+ or NG2+ (Figure 4D). We
observed a significant increase in the number of TUNEL+ NG2+
(11.2 ± 12.2 versus 1.9 ± 1.4 cells/section) and TUNEL+ GST-p+
(18.8 ± 15.2 versus 2.9 ± 5.3 cells/section) cells in the control group
compared to the FMD group (p < 0.05; Figures 4E and 4F). Taken
together, these results indicate that the FMD not only stimulates
regeneration and differentiation of oligodendrocytes but also pro-
tects OPCs and mature oligodendrocytes from apoptosis.
To investigate whether the FMD-dependent stimulation of
oligodendrocyte differentiation and remyelination can occur in-
dependent of the observed effects on T cell number and activity,
we used the cuprizone-induced demyelinating mouse model
(Ransohoff, 2012; Torkildsen et al., 2008). Addition of 0.2%
(w/w) cuprizone to the regular mouse diet for 5–6 weeks results
in demyelination in the corpus callosum followed by sponta-
neous remyelination upon re-feeding with regular chow. After
5 weeks of cuprizone treatment, mice were switched to either
the control diet or FMD cycles for 5 weeks, and some were
euthanized weekly to assess the degree of myelination by Luxol
fast staining and GST-p+ (Figures 4G and 4I). As expected, after
5 weeks of the cuprizone diet, a significant reduction in myelin
staining was observed in the corpus callosum compared to the
naive controls (Figures 4H and 4J). After two cycles, the FMD-
treated group displayed increased myelin staining and an
increased number of GST-p+ oligodendrocytes compared to
the control diet group (Figures 4H and 4J). However, at later
time points, we did not observe differences in spontaneous
re-myelination between the control diet and FMD cohorts, as it
is well established that cuprizone-dependent myelin damage
can be fully reversed after removal of the toxin (Figures S3C
and S3D). These results indicate that the FMD promotes OPC-
dependent regeneration and accelerates OPC differentiation
into oligodendrocytes while enhancing remyelination indepen-
dently of its modulation of the inflammatory response.
A Randomized Pilot Trial to Test the Effects of a FMD or
KD in Relapsing-Remitting MS Patients: Evidence for
Safety and Feasibility
A randomized, parallel-group, three-arm pilot trial (NCT01538355)
was conducted to assess the safety and feasibility of FMD or KD
treatment on health-related quality of life (HRQOL) in RRMS pa-
tients. 60 patients were randomly assigned to a control diet (CD;
n = 20), KD for 6 months (n = 20), or a single cycle of a modified
human FMD for 7 days (n = 20) followed by a Mediterranean diet
for 6 months (Figure S4). Baseline characteristics were balanced
among the three groups (Tables S1 and S2). The FMD and KD co-
horts displayed clinically meaningful improvements in the HRQOL
summary scales at 3 months, which included the overall quality of
life (Figure 4K) change in health (Figure 4L), a physical health com-
posite (Figure 4M), and a mental health composite (Figure 4N).
Also, similar changes were observed in the total HRQOL scales
at different time points (Figure S5). Adverse events (AEs) and
serious adverse events (SAEs) were reported for 92% (8%) of
CD cohort individuals, 78% (16%) of FMD cohort individuals,
and 78% (11%) of KD cohort individuals (Table S5). The most
common AE was airway infection, and the most frequent SAE
was lower urinary tract infection. No indication of an increase in
liver enzymes exceeding the normal range was observed in any
of the three treatment groups. Also, the interventions were well
tolerated, as evidenced by high compliance rates (CD, 60%;
KD, 90%; and FMD, 100%). During the 6-month study period,
we observed a total of eight relapses: four in the CD group, one
in the KD group, and three in the FMD group. In addition to
increased b-hydroxybutyrate levels in plasma, we observed a
slight reduction in lymphocytes and WBC counts and detected
a mild reduction in expanded disability status scale (EDSS) scores
in the FMD and KD groups (measured at baseline, month 3, and
month 6; Tables S1 and S6). Thus, there was an inverse associa-
tion between EDSS and HRQOL scores (Table S7). In MS patients
the FMD treatment lead to an over 20% drop in the total lympho-
cyte count (baseline versus day 8; Table S4) in 72% of the patients
(13 of 18 FMD-treated patients). WBC counts returned to the
baseline levels after these patients were switched to the Mediter-
ranean diet (month 3). Based on the mouse studies, these results
raise the possibility that the FMD alleviated symptoms in MS
patients by reducing the number of autoimmune lymphocytes.
Overall, our study indicates that the administration of FMD and
KD is safe, feasible, and potentially effective, but further studies,
including analyses such as magnetic resonance imaging (MRI),
blinded clinical assessments, and immune assays, are required
to determine efficacy.
DISCUSSION
An FMD administered every week was effective in ameliorating
EAE symptoms in all mice and completely reversed disease pro-
gression in a portion of animals after the onset of EAE signs. By
contrast, the KD had more modest effects and did not reverse
EAE progression in mice. FMD cycles appear to be effective in
the treatment of EAE in mice by (1) promoting oligodendrocyte
precursor-dependent regeneration and (2) reducing the levels
of microglia/monocytes and T cells contributing to autoimmunity
and encephalomyelitis. Our results support an FMD-mediated
anti-inflammatory effect possibly involving the upregulation of
AMPK or the downregulation of mTORC1, which sense nutrient
availability and dictate cell fate (Laplante and Sabatini, 2012). It
was shown that mTORC1 couples immune signals and metabolic
programming to establish Treg cell function (Zeng et al., 2013). In
fact, treatment with the mTORC1 inhibitor rapamycin or the
AMPK activator metformin attenuates EAE symptoms by modu-
lating effector T cells and Treg cells and restricting the infiltration of
mononuclear cells into the CNS (Esposito et al., 2010; Nath et al.,
2009). Therefore, FMD treatment could interfere with T cell prolif-
eration and differentiation and with recruitment of other immune
cells, resulting in a decreased recruitment at lesion sites (Fig-
ure 5). Some of these effects of the FMD may be triggered by
endogenous glucocorticoid production. Glucocorticoids are
used to treat MS relapses, but they are generally administered
in short bursts, since they can cause AEs such as osteoporosis
and metabolic syndrome (Brusaferri and Candelise, 2000; Ce
et al., 2006; Roth et al., 2010; Uttner et al., 2005). The FMD may
avoid these adverse effects by promoting additional and coordi-
nated endogenous responses. Importantly, FMD cycles also acti-
vated OPCs, resulting in myelin regeneration, as demonstrated
Cell Reports 15, 2136–2146, June 7, 2016 2143
Figure 5. A Simplified Model of FMD-Mediated Effects on Immune Suppression, Oligodendrocyte Regeneration, and Differentiation in MS
FMD treatment promotes endogenous glucocorticoid production, increases Treg cell numbers, blocks T cell activation, and promotes T cell death. In the lesion
area, FMD treatment reduces autoimmune T cell and microglia infiltration and promotes oligodendrocyte-precursor-dependent regeneration and differentiation
of myelinating oligodendrocytes, which engage with demyelinated axons to promote the formation of myelin sheaths.
by accelerated remyelination rate in the cuprizone model (Fig-
ure 5). Notably, because it is the alternation of FMD cycles and
re-feeding and not the FMD alone that promotes the regeneration
and replacement of autoimmune cells with naive cells, the use of
chronic restriction or even a chronic KD may not be effective, or
as effective, in the treatment of EAE and MS.
Finally, we report that the administration of the FMD and
KD in MS patients was safe and well tolerated and resulted
in high compliance. We observed positive effects of FMD cy-
cles or KD treatment in RRMS based on changes in self-re-
ported HRQOL and a mild improvement in EDSS (Table S6).
However, the lack of a proper Mediterranean diet control
makes it difficult to establish whether FMD cycles alone
are sufficient to produce these effects. In addition, MRI ana-
lyses and adequately blinded clinical assessments (EDSS
and multiple sclerosis functional composite [MSFC]), as well
as immune function analyses would greatly enhance the
strength of the clinical findings. Because, unlike for the mouse
experiments, the FMD was only administered to patients only
once, it will be important to test the effects of multiple FMD
cycles on MS patients in larger, randomized, and controlled
trials.
EXPERIMENTAL PROCEDURES
EAE Model
C57Bl/6 (10-week-old female) mice were purchased from The Jackson Lab-
oratory and immunized subcutaneously with 200 mg MOG35–55 (GenScript)
2144 Cell Reports 15, 2136–2146, June 7, 2016
mixed 1:1 with supplemented complete Freund’s adjuvant followed by
200 ng pertussis toxin (PTX; List Biological Laboratories) intraperitoneally
(i.p.) at days 0 and 2. For adoptive transfer, spleens from active immunized
mice were isolated and red blood cells (RBCs) were lysed. Spleen cells were
cultured in the presence of MOG35–55 (20 mg/ml) with rmIL-23 (20 ng/ml) for
48 hr. Cells were collected and re-suspended in PBS, and 15 million cells
were injected intravenously. See Supplemental Experimental Procedures
for a detailed description of disease severity scoring. All experiments
were performed in accordance with approved Institutional Animal Care
and Use Committee (IACUC) protocols of the University of Southern
California.
Mouse Fasting Mimicking Diet
Mice were fed ad lib with irradiated TD.7912 rodent chow (Harlan Teklad),
containing 15.69 kJ/g digestible energy (animal-based protein 3.92 kJ/g,
carbohydrate 9.1 kJ/g, and fat 2.67 kJ/g). The experimental FMD is based
on a nutritional screen that identified ingredients that allow high nourish-
ment during periods of low calorie consumption. The FMD diet consists
of two different components, day 1 diet and day 2–3 diet, that were fed
in this order, respectively. See Supplemental Experimental Procedures
for a detailed explanation of the FMD. Mice consumed all the supplied
food on each day of the FMD regimen and showed no signs of food aver-
sion. After the end of FMD, we supplied TD.7912 chow ad lib for 4 days
before starting another FMD cycle. Prior to supplying the FMD, animals
were transferred into fresh cages to avoid feeding on residual chow and
coprophagy.
Clinical Trial Design
This study was a three-armed, parallel-group, single-center, controlled,
and randomized clinical pilot trial to assess the effects of dietary interven-
tions on HRQOL in RRMS patients. The permuted-block randomization
was generated online at http://randomization.com. An investigator blind
to the randomization plan determined the patients’ randomization number
before they underwent the randomization step. This study is registered at
http://www/clicaltrials.gov as NCT01538355. The study was approved by
the local ethics committee. All participants gave informed written consent
according to the 1964 Declaration of Helsinki. See Supplemental Experi-
mental Procedures for detailed descriptions of the clinical trial and diet
compositions.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and seven tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2016.05.009.
AUTHOR CONTRIBUTIONS
I.Y.C., L.P., M.W., and V.D.L. designed mouse experiments. I.Y.C., S.B., and
P.C. performed the mouse experiment. I.Y.C., L.P., P.C., B.B., and A.G.
performed and processed immunohistochemistry. I.Y.C., L.P., and B.B. per-
formed qualitative and quantitative analysis. I.Y.C. and J.S. performed fluores-
cence-activated cell sorting (FACS) analysis. I.Y.C. processed the cytokine
assay. A.M., F.P., and M.B. designed the human study; M.B. acquired human
clinical data; A.M., F.P., and M.B., analyzed and interpreted data; and M.B.
performed, interpreted, and presented the statistical analysis. A.M., F.P.,
M.B., A.H.C., T.E.M., M.W., and V.D.L. were involved in discussing the results
and editorial support. I.Y.C., M.B., and V.D.L. wrote the paper. All authors
discussed the results and commented on the manuscript.
CONFLICTS OF INTEREST
The University of Southern California has licensed intellectual property to
L-Nutra that is under study in this research. As part of this license agreement,
the University has the potential to receive royalty payments from L-Nutra.
V.D.L. has equity interest in L-Nutra, a company that develops medical food.
ACKNOWLEDGMENTS
We thank Dr. Stephen Hauser for insightful comments, Dr. Pinchas Cohen for
assistance with fluorescence microscopy, and Nadine Krueger and Gabi Rahn
for technical assistance. L.P. is a Harry Weaver Neuroscience Scholar of the Na-
tional Multiple Sclerosis Society (NMSS, JF 2144A2/1) and is funded by Fonda-
zione Italiana Sclerosi Multipla (FISM; 2014/R/15) and the Office of the Assistant
Secretary of Defense for Health Affairs, through the Multiple Sclerosis Research
Program, under award number W81XWH-14-1-0156. The opinions, interpreta-
tions, conclusions, and recommendations express in this article are those of
the author and are not necessarily endorsed by the Department of Defense.
The mouse study was funded by National Institutes of Health (NIH)/National Insti-
tute on Aging (NIA) grant AG034906 (to V.D.L.). The human study was funded by
Meylin Projekt e.V. and Familie Ernst Wendt Stiftung Stadt Koeln, which were not
involved in any decision-making processes relating the study or its participants.
The work of F.P. is supported by Deutsche Forschungsgemeinschaft (DFG Exc
257). The content is solely the responsibility of the authors and does not neces-
sarily represent the official views of the NIA or NIH. The University of Southern Cal-
ifornia has licensed intellectual property to L-Nutra that is under study in this
research. As part of this license agreement, the university has the potential to
receive royalty payments from L-Nutra. V.D.L. has equity interest in L-Nutra, a
company that develops medical food.
Received: May 22, 2015
Revised: February 20, 2016
Accepted: April 26, 2016
Published: May 26, 2016
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Cell Reports, Volume 15

Supplemental Information

A Diet Mimicking Fasting Promotes

Regeneration and Reduces Autoimmunity
and Multiple Sclerosis Symptoms
In Young Choi, Laura Piccio, Patra Childress, Bryan Bollman, Arko Ghosh, Sebastian
Brandhorst, Jorge Suarez, Andreas Michalsen, Anne H. Cross, Todd E. Morgan, Min
Wei, Friedemann Paul, Markus Bock, and Valter D. Longo
Supplemental Figure 1
a b c d

Serum Glucose (mg/dL)

120 EAE-CTRL 600 300 2.5 ***
* Naive Naive Naive

Serum IGF-1 (ng/mL)

EAE-FM D

OH Butyrate (mM)

110 EAE-KD EAE-CTRL * EAE-CTRL 2.0 EAE-CTRL
EAE-FMD EAE-FMD EAE-FMD
400 200
BW (%)

100 1.5

90 1.0
200 100
80 0.5

70 0 0 0.0
0 5 10 15 20 25 30 D3

ve
D3 D14

e
D14

ve
D3 D14

v
Days after Initial Symptoms

ai
ai

ai
N
N

N
e f g
4 *** *** *** 50 EAE-CTRL

(# of Positive Pixels/mm 2)
EAE-CTRL
Mean clinical EAE score

EAE-FMD 5 EAE-FMD
EAE CTRL 40
Mean Clinical EAE Score

3
EAE Caloric Restricted
4
30

MBP
2 3

2
20
1
1 10

0 0 0
D3 D14 D30 0 5 10 15 20
D14

EAE CTRL EAE FMD EAE CTRL EAE FMD

h

D3 D30
k **
l **
m n
3 3 40 0.8
EAE-CTRL EAE-CTRL
(# of Positive Pixels/mm 2)

EAE-CTRL
(# of Positive Pixels/mm 2)

EAE-CTRL
Degree of Demyelination
Inflammatory Infiltration

EAE-FMD EAE-FMD EAE-FMD ** EAE-FMD

30 0.6
2 2
SMI32
(a.u.)

(a.u.)

MBP

20 0.4 *
1 1
** ** 10 0.2

0 0 0 0.0
D3 D30 D3 D30 D3 D30 D3 D30
Figure S1. The beneficial effect of MOG-induced EAE disease progression persist a long
term. Related to Figure 1
a. Body weights of control mice (Black), mice fed with ketogenic diet (Red), and mice fed with
FMD-Thr (Blue) (% BW ± S.E.M.).
b. Serum IGF-1 (ng/mL) level of naive, EAE Day1 before treatment, CTRL and FMD at Day3
and D14 (mean± S.E.M.; p < 0.05; t-test).
c. Serum glucose (mg/dL) level of naive, EAE Day1 before treatment, CTRL and FMD at
Day3, Day7 and D14 (mean± S.E.M.; p < 0.05; t-test).
d. Serum ketone body-βOH Butyrate (mM) level of naive, CTRL and FMD at Day3 and D14.
e. Average clinical score at different time points 3 days, 14 days, or 30 days post initial sign
(n=23; *** p < 0.001; t-test).
f. EAE severity score of control diet (EAE CTRL), and the same dietary composition of the
control diet but matching the calories of the FMD (EAE CR; n=6)] (mean ± S.E.M.).
g. Quantification of MBP of EAE CTRL and EAE FMD on Day14 (n = 8, mean ± S.E.M.).
h-n. The representative staining (h-j) and quantification (k-n) of (h) H&E, (i) solochrome
cyanide, (j) MBP and SMI32 at D3 and D30 of control and FMD (n = 8, mean ± S.E.M., * p <
0.05, ** p<0.01; t-test; Scale bar represents 200 µm).
 i
a

l
c
Serum TNF (pg/mL) % of WBC
% of CD4+ Splenocyte

0
10
20
30
40
50
0
50
100

0
50
100
150
CD62L N
ai
EA v
E- e
C
TR
EA L

CD44
Naive EAE
E-
FM
D

p=0.09

j
EAE CTRL
BrdU + (% of live cells)

FMD

*
0
5
10
15
20
EA
E
C
TR
L

d
FMD-RF
EA
E % of CD8 + Splenocyte
Supplemental Figure 2

FM

0
5
10
15
20

p=0.07
D N
ai
EA v

NAIVE
E- e
C
Mon%
Gran%

TR

EAE-FMD
Lymph%

EAE-CTRL
EA
E-

EAE FM D

k
FM
D

m
Serum IFN  (pg/mL) CD4+ IL17+
b

F4/80 + Cells
e

(% of BrdU) % of CD8 +

g
% of CD4 + CD44 H

0
25
50
75
100
0
25
50
75
100
0
5
10
15
20

0
200
400
600
800
1000
0
2
4
6
EA EA
E N N E
C EA ai
v EA ai
v C
TR TR
L E- e E- e L
C C

*
EA TR TR EA
E
EA L EA L E
FM E- E- FM
D FM FM D
D D

% of CD8 + CD44 H

0
25
50
75
h 100
CD44 L

N
CD44 H

ai
v
EAE FMD

EA
EAE CTRL

E- e
C
TR

NAIVE
EA L

EAE-FMD
E-

EAE-CTRL
FM
D

n
Serum IL-17 (pg/mL)

0
10
20
30
40
50
*
CD62L low
CD62L high

NAIVE

EAE-FMD
EAE-CTRL
Figure S2. FMD modulate immune response by reducing CD4 T cell activation. Related to
Figure 2
a. % Lymphocyte, monocyte, and granulocyte of total white blood cell of Naïve, EAE CTRL,
EAE FMD, and EAE FMD-RF (n=4-6; Mean ± S.E.M.).
b. Quantification of F4/80+ cells (% of total splenocyte) of EAE CTRL and EAE FMD (n=4;
mean ± s.e.m.).
c. % of CD3+ CD4+ splenocyte of control or FMD. FMD treatment had no significant
difference in % of CD3+ cells (n=4; mean ± s.e.m.).
d. % of CD3+ CD8+ splenocyte of control or FMD. FMD treatment had no significant
difference in % of CD3+ cells (n=4; mean ± s.e.m.).
e. CD8+ splenocyte activation level (CD8+CD44High) of naïve, CTRL and FMD (n=4; mean ±
s.e.m.).
f. Representative flow cytometry plot of CD44 and CD62L and quantification of EAE CTRL
and EAE FMD (n=4; mean ± s.e.m.).
g-h. % ratio of effector (CD44High and CD62Llow) to memory T-cells (CD44High and CD62LHigh)
population from (g) CD4+CD44high T cells, and (h) CD8+CD44high T cells (n=4, mean ± s.e.m.).
i. BrdU injection timeline. BrdU was given during the re-feeding period (4 injections within 48
hours, 1 mg of BrdU / injection).
j. Quantification of BrdU+ lymphocytes of EAE CTRL and EAE FMD (n=4, mean ± s.e.m.).
k. Quantification of CD4+IL17+ BrdU+ lymphocytes of EAE CTRL and EAE FMD (n=4, mean ±
s.e.m.).
l-n. Serum levels of (l) TNF-α, (m) IFN-γ, and (n) IL-17 at Day25
Supplemental Figure 3

a b c
600 ** 1500 ** ** 200

TUNEL + Cells / section

# NG2+ Cells / section

Naive *
EAE-CTRL

# GST/mm2
150
400 EAE-FMD 1000

100

200 500
50
*
0 0 0
D3 D14 D3 D14

D
TR

FM
C

E-
E-

EA
EA
d e
*** ***
Naive Naive
Luxol Fast Staining

100 100 *
Cup (5 weeks) Cup (5 weeks)
GST + / DAPI in CC

**
% Ctrl Pixel

Cup+CTRL Cup + CTRL

(%) of Naive

75 Cup+FMD 75 Cup + FMD

50 50

25 25

0 0
Week1 Week3 Week5 Week1 Week3 Week5
Weeks after Cuprizone Withdrawal Weeks after Cuprizone Withdrawal

Figure S3. FMD increases oligodendrocyte differentiation and protection of OPC and
oligodendrocytes. Related to Figure 4
a. Quantification of number of NG2+ (oligodendrocyte precursor cells) at D3 and D14.
b. Quantification of number of GSTπ+ (matured oligodendrocyte) at D3 and D14.
c. Quantification of number of TUNEL+ cells sowing a significantly increased number of
apoptotic cells in control but not in FMD on D3 (n=6; *p<0.05; t-test).
d. Luxol fast staining quantification (% Naïve control pixel) of naïve mice, cuprizone fed (5
weeks) mice, control diet fed mice and FMD cycle fed mice at week 1, week3 and week
5 upon withdrawal of cuprizone diet.
e. GSTπ quantification (% Naïve of # of GSTπ+ / DAPI+ in corpus callosum) of naïve mice,
cuprizone fed (5 weeks) mice, control diet fed mice and FMD cycle fed mice at week 1,
week3 and week 5 upon withdrawal of cuprizone diet.
Supplemental Figure 4

Figure S4. Schematic diagram displaying the time course of clinical trial and the diet
interventions. Related to Figure 4
A randomized parallel-group 3 arm pilot trial (NCT01538355) was with relapsing-
remitting MS patients. 60 patients were randomly assigned to: control diet (CD), KD for 6
months or a single cycle of modified human FMD for 7 days followed by a Mediterranean diet
for 6 months
 j
a

p
g
d

m
Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline
Mean Change from Baseline
Emotional Well-Being (MS-54) Sexual Function (MS-54) Overall Quality of Life (MS-54) Energy / Fatigue (MS-54) Physical Health Composite (MS-54)
Satisfaction Sexual Function (MS-54)

10
15
20

0
5
-5
10
15
20
-10
-5
10
15
10
15
20

0
5
0
5
0
5
10
15

0
5
KD
CD

-10
0
10
20
30
FMD

1
*

1
1
1
1

3
*

M onth
M onth

3
*

3
3
3

M onth
M onth
M onth
M onth
Supplemental Figure 5.

6
6

6
6
6
6

k
e

n
h
b

Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline
Role Limitations Emotional (MS-54) Social Function (MS-54) Change in Health (MS-54) Role Limitations Physical (MS-54) Mental Health Composite (MS-54)

-10
-5
0
5
10
15
-20
-10
0
10
20
30

-20
-10
0
10
20
30
-40
-30
-20
-10
0
10
20
30
40
-5
0
5
10
15

1
1
*

1
1
*
*

3
*

3
*

M onth
3
3
*
Month
M onth

M onth
M onth
*

6
*

6
6

6
6
*

l
i
f
c

o
Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline Mean Change from Baseline
Cognitive Function (MS-54) Health Distress (MS-54) Health Perception (MS-54) Bodily Pain (MS-54) Physical Function (MS-54)

-10
-10

-5
0
5
10
15
-5
0
5
10
15
-10
-5
0
5
10
15
-10
0
10
20
30
-15
-10
-5
0
5
10
15

1
1
1
1
1

*
*

3
3
3
3
3

M onth
M onth
M onth
M onth

M onth
*
*

6
6
6
6
6
*
Figure S5. The MS-54 scores at Month 1, Month3 & Month6. Related to Figure 4
This pilot clinical feasibility trial revealed potentially positive effects on HRQOL based on self-
reports for both FMD and KD. Mean change from baseline of CD, FMD, and KD at month 1, 3
and 6 of physical health composite (a), mental health composite (b), physical function (c),
energy/fatigue (d), role of limitation physical (e), bodily pain (f), overall quality of life (g),
change in health (h), health perception (i), sexual function (j), social function (k), health distress
(l), emotional well-being (m), role limitations emotional (n), cognitive function (o), and
satisfaction sexual function (p) are shown. Dotted line represents threshold which is thought to
be clinically important (≥ 5 points) in MS-54 outcome. FMD was performed only once which
resulted in a maximum effect size at month 3; thus study time between Month 3 and 6 is
suggested to be a washout period of FMD treatment (mean ± SED; * p<0.05; Mann-Whitney-U
test. Increase of ≥ 5 points are considered as clinically important). At month 3 and 6, the CD had
negligible effect sizes (0.04 to 0.08) and no clinically meaningful impact (mean change from
baseline (MCB) > 5) (Norman et al., 2003, Rudick et al., 2007, Kappos et al., 2014) on physical
health composite (PHCS; 0.22+11.4; MCB + SD) and on mental health composite (MHCS;
1.88+19.9) and on most sub-scales of the MS-54 (Supplemental Fig. 5; Supplemental Table
3). In contrast, patients in the FMD group showed clinically meaningful improvement on MS-54
scores with medium to large effect sizes (0.4 to 0.5) after 3 months, including increases on PHCS
(7.27+4.3), MHCS (8.85+14.2) and on 10 out of 14 of sub-scales (Supplemental Fig. 5;
Supplemental Table 3). After 6 months, the KD group showed medium to large effect sizes (0.3
to 0.5) and clinically meaningful improvement in PHCS (8.37+11.0), MHCS (6.27+14.8) and on
11 out of 14 of sub-domains (Fig. 4k-n; Supplemental Fig. 4; Supplemental Table 3).
Supplementary Table 1. Summary of demographics and primary outcome at baseline.
Related to Figure 4 and Supplementary Figure 4.
Supplementary Table 2. Summary of secondary outcome parameters at baseline. Related
to Figure 4, Supplementary Figure 4 and 5.
Supplementary Table 3. Mean change from baseline (MCB) in Multiple Sclerosis Quality
of Life (MS-54) scores. Related to Figure 4 and Supplementary Figure 5.
Supplementary Table 4. Mean Change from Baseline (MCB) in white blood cells and
lymphocytes. Related to Figure 4 and Supplementary Figure 5.
Supplementary Table 5. Adverse events and safety parameters. Data are number of events
(number of individuals). Related to Supplementary Figure 5.
Supplementary Table 6. Change from baseline in expanded disability severity scales
(EDSS). Related to Supplementary Figure 5
Supplementary Table 7. Correlation analysis between baseline MS-54 scores and EDSS
scores. Related to Supplementary Figure 5.
Supplementary Table 1: Summary of demographics and primary outcome at baseline. Data are mean (SD), number (%) or median (inter quartile range).
Baseline data of secondary outcomes are given in supplementary table 1. *Kruskal Wallis test for comparison between the three groups was performed.
Baseline data were available for 48 patients deviations are given in brackets (n=control diet, fasting mimicking diet, ketogenic diet).

Total CTRL FMD KD Diet *p-

SD IQR SD IQR SD IQR SD IQR
(n=48) (n=12) (n=18) (n=18) value
Baseline characteristics
Age in years 44.8 10.4 50.5 10.4 44.4 11.1 41.3 8.2 0.0505
38/10 9/3 15/3 14/4
Gender F/M (79/21) (75/25) (83/17) (78/22) 0.848
Expanded disability status score 3 2.0-4 2.5 1.5-4 4 2.4-4 3 2.4-3.5 0.2305
Disease Duration in years 8.9 7.3 9.9 9.2 11 7.7 6.3 4.3 0.0882
Relapse rate 12 months prior study outset 0.4 0.5 0.33 0.65 0.39 0.5 0.44 0.5 0.6849
No immune modulating drugs 11 (23) 3 (25) 2 (11) 6 (33) 0.3

Glatirameracetate 15 (31) 7 (58) 6 (33) 2 (11) 0.025

Interferon beta 1a 9 (19) 1 (8) 6 (33) 2 (11) 0.1

Interferon beta 1b 3 (6) 0 1 (6) 2 (11) 0.5

Fingolimod 4 (8) 0 1 (6) 3 (17) 0.2

Natalizumab 4 (8) 1 (8) 1 (6) 2 (11) 0.8

Intravenous immuneglobulin 2 (4) 0 1 (6) 1 (6) 0.7
BMI 26.7 5.5 27.3 6.9 26 4.8 26.9 5.3 0.9047
Weight Kg 78 17.9 80.2 22.7 74.5 13.9 80 18.4 0.5792
Percent Body Fat 36.6 10.4 38.03 10.57 35.73 9.912 36.49 11.27 0.8568

Primary outcome parameters

Physical Health Composite (n=12,13,13) 67.4 15.2 73.09 8.786 59.61 15.55 69.93 17.19 0.0701
Mental Health Composite (n=12,17,15) 71.1 16.8 75.42 13.56 64.21 19.08 75.47 14.71 0.0925
Physical Function (n=12,18,16) 75.9 25.5 87.9 18.4 67.4 28.81 76.5 23.76 0.0636
Health Perception (n=12,17,17) 54.8 19.9 57.5 16.86 49.9 16.68 57.7 17 0.2738
Energy/Fatigue (n=12,17,17) 46.2 17.3 52.33 19.18 40.47 15.48 47.53 16.79 0.2425
Role Limitations Physical (n=12,18,15) 58.6 37.5 85.42 24.91 42.36 28.16 56.67 44.79 0.0072
Pain 72.7 25 83.2 21.9 59.4 25.3 79 21.7 0.0187
Sexual Function (n=12,14,16) 74.6 27.2 59.68 32.13 79.78 21.36 81.26 25 0.1479
Social Function 79.2 18.8 80.56 16.41 79.39 18.76 78.22 21.05 0.9897
Health Distress (n=12,18,16) 73.3 18.1 75.03 11.91 64.78 22.07 81.68 12.6 0.0619
Overall Quality of Life 67.9 15.6 75.51 13.13 60.11 16.46 70.65 13.28 0.0262
Emotional Well Being (n=12,17,17) 69.5 17.2 71.67 18.25 64.47 19.89 72.94 12.77 0.4105
Role Limitations Emotional (n=12,18,17) 75.2 37.1 80.56 26.44 64.81 44.97 82.36 33.57 0.455
Cognitve Function (n=12,18,16) 70.2 18.2 74.75 13.62 68.4 19.35 68.75 20.16 0.5023
Change in Health 47.9 18.5 50 10.66 41.67 19.17 52.78 20.81 0.2247
Satisfaction with Sexual Function
(n=12,16,16) 46.2 31.3 40.28 31.36 44.78 33.74 52.09 29.75 0.5493
Supplementary Table 2
Summary of secondary outcome parameters at baseline. Data are mean (SD). *Kruskal Wallis test for comparison
between the three groups was performed. Baseline data were available for 48 patients deviations are given in brackets
(n=control diet. fasting mimicking diet. ketogenic diet).

SD Control Fasting
Total SD Ketogenic SD
Diet SD IQR Mimicking *p-value
(n=48) IQR IQR Diet (n=18) IQR
(n=12) Diet (n=18)
Baseline characteristics

RR systolic 120.8 15.5 121.3 16.5 125.5 16 115.7 13.3 0.1334

RR diastolic 72.4 9.1 70 13 73.9 6 72.3 9 0.6298

Energy intake in kcal per day

(n=9,12,12) 1686 456 1912 340 1484 413 1718 514 0.1048

Carbohydrate intake per day in

g (n=9,12,12) 179.1 59.8 208.4 70.8 163.5 50.5 172.7 56.4 0.2465

Fat intake per day in g

(n=9,12,12) 68.5 20.9 76.3 16.3 59 17.4 72.1 24.8 0.1305

Protein intake per day in g

(n=9,12,12) 72.6 27.8 79.2 37.4 62.8 22.36 77.31 23.69 0.3136

Beck Depression Inventory

(n=10,15,17) 9.0 6.8 7.6 6.2 11.3 7.7 7.8 6.1 0.2463

Fatigue Severity Scale 4.3 1.7 3.6 1.9 5.2 1.1 3.9 1.8 0.0235

Modified Fatigue Impact Scale

(n=9,16,18) 33.1 17.6 24.1 19.2 38.3 15.7 32.9 17.5 0.2474

Visual Analogue Scale Fatigue

(n=12,16,18) 4.0 2.3 3.6 2.2 4.1 2.5 4.1 2.3 0.7991

Multiple Sclerosis Functional

Composite 3 z-score 0.16 0.69 -0.081 0.7 -0.027 0.89 0.431

Multiple Sclerosis Functional

Composite 2 z-score 0.29 0.74 -0.099 0.64 -0.093 0.88 0.0906

Timed 25 foot Walk Test in sec. 6.0 6.2 5.7 4.7 5.5 2.1 6.6 9.4 0.3019

9-hole peg test in sec. 21.8 7.2 20.8 3.0 21.9 4.700 22.5 8.1 0.8745

Paced Auditory Serial Addition

Test 3 45.1 10.7 48.6 10.1 42.4 11.7 45.5 10 0.2689

Paced Auditory Serial Addition

Test 2 33.0 9.9 39.9 11.5 29.9 8.8 31.4 8 0.0371
ALT - U/l 23.6 14.6 20.7 8 26.9 18.3 22.2 13.9 0.6689

AST - U/l 24.2 8.4 24.5 7.3 25.1 8.6 23.2 9.2 0.6862

y-GT U/l 27.2 38.7 20.9 13.8 36.1 60.2 22.6 16.4 0.804

White Blood Cells count/nl 6.451 2.266 6.097 1.17 6.523 2.742 6.614 2.381 0.8949

Lymphocytes count/nl 1.943 1.028 1.877 0.6592 1.912 1.142 2.018 1.152 0.9287
Supplementary Table 3
Mean change from baseline (MCB) in Multiple Sclerosis Quality of Life (MS-54) scores. Comparison for rates of intra- and inter-group change in MS-54
measures. We considered for statistical analysis results of patients with fully completed data sets.*Friedmann test for intra-group gradient. **Mann-Whitney-
U test for inter-group differences. Clinically relevant changes are indicated in bold.

Control Diet Fasting Mimicking Ketogenic Diet FMD - CD KD - CD

Diet (FMD)
(CD) (KD)
MS-54
Domains n MCB SD *p n MCB SD *p n MCB SD *p Differences (95%CI) **p Differences (95%CI) **p
Baseline 73.09 8.79 59.61 15.55 <0.05 69.93 17.19 Mean Lower Upper Mean Lower Upper
Physical
Health Month 1 12 1.33 6.05 13 3.44 8.86 13 2.43 8.61 2.11 -4.22 to 8.44 1.09 -5.11 to 7.30
Composite Month 3 12 0.42 8.37 13 7.27 7.52 13 4.93 13.92 6.85 0.28 to 13.42 <0.05 4.51 -5.10 to 14.12
(PHCS)
Month 6 12 0.22 11.41 13 4.33 8.81 13 8.37 11.02 4.11 -4.29 to 12.50 8.15 -1.13 to 17.43

Baseline 75.42 13.56 64.21 19.08 75.47 14.71 <0.05

Mental
Health Month 1 12 -0.05 7.03 17 9.65 13.66 15 0.59 9.35 9.69 1.69 to 17.69 <0.05 0.64 -6.07 to 7.35
Composite Month 3 12 1.15 14.38 17 8.85 14.16 15 3.46 14.18 7.70 -3.33 to 18.72 <0.05 2.31 -9.07 to 13.69
(MHCS)
Month 6 12 1.88 19.90 17 2.69 15.80 15 6.27 14.84 0.81 -12.8 to 14.42 4.39 -9.37 to 18.15

Baseline 87.90 18.40 67.40 28.81 76.50 23.76 <0.005

Physical Month 1 12 -1.25 6.44 18 -8.48 15.70 16 -0.87 13.28 -7.23 -15.8 to 1.32 0.38 -8.18 to 8.95
Function Month 3 12 -7.50 11.77 18 3.72 14.21 16 5.69 12.03 11.22 1.06 to 21.38 <0.05 13.19 3.84 to 22.55 <0.005

Month 6 12 -6.67 14.03 18 4.27 9.79 16 7.26 13.07 10.94 2.05 to 19.83 <0.05 13.92 3.34 to 24.51 <0.005

Baseline 57.50 16.86 49.90 16.68 57.70 17.00

Health Month 1 12 3.75 15.24 17 2.42 11.63 17 -2.65 12.55 -1.33 -11.6 to 8.90 -6.40 -17.0 to 4.20
Perception Month 3 12 0.42 13.39 17 0.40 13.36 17 3.97 18.86 -0.02 -10.4 to 10.33 3.55 -9.48 to 16.59

Month 6 12 6.57 13.51 17 0.36 12.53 17 6.62 15.54 -6.20 -16.2 to 3.81 0.05 -11.4 to 11.46

Baseline 52.33 19.18 40.47 15.48 <0.05 47.53 16.79 <0.01

Energy / Month 1 12 0.00 7.24 17 7.71 15.07 17 3.76 9.54 7.71 -0.97 to 16.39 3.76 -2.63 to 10.16
Fatigue Month 3 12 0.67 17.55 17 9.65 12.89 17 10.35 18.82 8.98 -2.59 to 20.56 9.69 -4.48 to 23.85

Month 6 12 4.23 23.87 17 1.41 13.78 17 10.82 19.79 -2.81 -17.2 to 11.55 6.60 -10.1 to 23.27

Baseline 85.42 24.91 42.36 28.16 <0.005 56.67 44.79

 -
Month 1 12 23.44 18 0.69 36.50 15 6.67 39.49 4.86 -19.6 to 29.30 10.83 -15.8 to 37.46
4.167
Role
Limitations Month 3 12 -2.08 29.11 18 28.47 37.33 15 8.33 48.80 30.56 4.34 to 56.77 <0.05 10.42 -22.5 to 43.37
Physical
-
Month 6 12 27.09 18 13.19 32.22 15 15.00 44.12 27.78 4.64 to 50.91 <0.05 29.58 -0.40 to 59.57
14.58

Baseline 83.20 21.90 59.40 25.30 <0.05 79.00 21.70

Month 1 12 3.18 9.68 18 12.68 27.23 18 5.37 19.56 9.51 -4.98 to 24.00 2.20 -10.3 to 14.71
Pain
Month 3 12 2.36 18.96 18 16.90 24.27 18 4.27 20.04 14.55 -2.51 to 31.60 <0.05 1.91 -13.1 to 16.89

Month 6 12 -0.84 20.26 18 11.19 20.94 18 6.40 17.81 12.03 -3.75 to 27.82 7.23 -7.1 to 21.60

Baseline 59.68 32.13 79.78 21.36 81.26 25.00

Sexual Month 1 12 5.63 17.38 14 1.79 10.41 16 -2.43 8.69 -3.83 -15.2 to 7.57 -8.06 -19.7 to 3.59
Function Month 3 12 8.61 18.39 14 -1.20 12.16 16 8.84 13.76 -9.81 -22.3 to 2.64 0.23 -12.2 to 12.70

Month 6 12 11.85 17.21 14 5.96 19.46 16 0.52 14.73 -5.89 -20.9 to 9.10 -11.3 -23.8 to 1.09

Baseline 80.56 16.41 79.39 18.76 78.22 21.05

Social Month 1 12 5.56 10.25 18 -2.77 21.15 18 5.11 15.42 -8.33 -20.2 to 3.59 -0.45 -10.8 to 9.95
Function Month 3 12 4.17 8.35 18 2.55 20.59 18 6.49 20.10 -1.61 -14.5 to 11.27 2.33 -10.3 to 14.93

Month 6 12 1.38 12.74 18 4.41 17.29 18 7.43 12.74 3.03 -8.93 to 14.98 6.05 -3.68 to 15.77

Baseline 75.03 11.91 64.78 22.07 81.68 12.60

Health Month 1 12 -0.54 16.76 18 8.84 14.88 16 1.941 12.44 9.38 -2.56 to 21.33 2.27 -8.35 to 12.88
Distress Month 3 12 3.09 15.88 18 5.48 14.45 16 4.662 16.05 2.39 -9.08 to 13.86 1.05 -10.7 to 12.84

Month 6 12 4.15 16.50 18 5.72 22.14 16 6.234 14.94 1.57 -13.8 to 16.93 2.08 -10.2 to 14.34

Baseline 75.51 13.13 60.11 16.46 <0.05 70.65 13.28

Overall Month 1 12 -4.36 13.78 18 7.88 9.98 18 0.80 16.71 12.24 3.37 to 21.11 <0.05 5.16 -6.76 to 17.08
Quality of
Life Month 3 12 -1.33 8.37 18 10.87 11.48 18 3.53 14.61 12.20 4.28 to 20.11 <0.005 4.86 -4.71 to 14.43

Month 6 12 -0.86 14.58 18 5.51 12.85 18 5.03 17.47 6.37 -3.98 to 16.71 5.89 -6.62 to 18.41

Baseline 71.67 18.25 64.47 19.89 72.94 12.77

Emotional Month 1 12 2.67 7.69 17 11.53 17.94 17 2.59 12.40 8.86 -1.24 to 18.96 -0.08 -8.38 to 8.23
Well-
Being Month 3 12 1.67 15.77 17 7.53 17.54 17 3.18 18.93 5.86 -7.17 to 18.89 1.51 -12.2 to 15.21

Month 6 12 3.25 21.34 17 4.94 13.23 17 5.53 16.49 1.69 -11.5 to 14.85 2.28 -12.1 to 16.68
 Baseline 80.56 26.44 64.81 44.97 82.36 33.57
Role Month 1 12 0.00 28.43 18 12.97 38.17 17 -7.85 32.34 12.97 -13.5 to 39.43 -7.85 -31.7 to 15.98
Limitations
Emotional Month 3 12 2.78 33.23 18 12.97 34.57 17 3.92 28.58 10.19 -15.8 to 36.19 1.14 -22.5 to 24.79

Month 6 12 2.78 41.33 18 -1.85 44.99 17 -3.93 48.42 -4.63 -37.9 to 28.65 -6.70 -42.0 to 28.62

Baseline 74.75 13.62 68.40 19.35 68.75 20.16

Cognitive Month 1 12 0.25 7.34 18 2.08 15.46 16 2.73 15.97 1.83 -8.01 to 11.68 2.48 -6.88 to 11.85
Function Month 3 12 -1.31 12.88 18 5.21 8.64 16 3.25 16.28 6.52 -1.51 to 14.55 4.57 -7.16 to 16.29

Month 6 12 -1.05 15.95 18 2.43 15.77 16 7.03 16.59 3.49 -8.61 to 15.58 8.08 -4.73 to 20.90

Baseline 50.00 10.66 41.67 19.17 0.01 52.78 20.81 <0.05

Change in Month 1 12 -2.08 16.71 18 12.50 23.09 18 11.11 26.04 14.58 -1.31 to 30.48 <0.05 13.19 -4.24 to 30.63
Health Month 3 12 -4.17 20.87 18 15.28 22.91 18 16.67 30.92 19.44 2.55 to 36.34 0.01 20.83 -0.09 to 41.76 <0.05

Month 6 12 -2.08 19.82 18 13.89 19.60 18 14.58 29.78 15.97 0.94 to 31.00 <0.05 16.67 -3.43 to 36.76

Baseline 40.28 31.36 44.78 33.74 52.09 29.75

Satisfaction Month 1 12 -3.47 10.92 16 7.29 21.92 16 0.9375 15.62 10.77 -2.31 to 23.85 4.41 -6.45 to 15.27
with Sexual
Function Month 3 12 6.93 21.87 16 15.63 31.91 16 0.01 21.09 8.70 -13.4 to 30.76 -6.93 -23.7 to 9.89

Month 6 12 4.15 28.56 16 14.59 30.36 16 3.14 27.37 10.44 -12.8 to 33.68 -1.01 -22.9 to 20.87
Supplementary Table 4

Mean Change from Baseline (MCB) in white blood cells and lymphocytes. Comparison for rates of intra- and inter-group change in outcome measures. *
Wilcoxon matched-pairs signed rank test for intra-group comparison. **Mann-Whitney-U test for inter-group differences.

Control Diet (CD) Fasting Mimicking Ketogenic Diet (KD) FMD - CD KD - CD

Diet (FMD)
Laboratory
parameters n MCB SD *p n MCB SD *p n MCB SD *p Differences (95%CI) **p Differences (95%CI) **p

White Blood Baseline 6.10 1.17 6.52 2.74 6.61 2.38 Mean Lower Upper Mean Lower Upper
Cells
Month 1 12 0.19 1.22 18 -0.94 2.13 0.08 18 -0.44 1.42 -1.13 -2.53 to 0.27 0.09 -0.63 -1.66 to 0.40
count/nl
Month 3 12 0.49 1.21 18 -0.18 1.73 18 -0.27 1.95 -0.67 -1.85 to 0.52 -0.76 -2.06 to 0.53

Month 6 12 0.81 1.77 18 -0.48 2.06 18 -0.72 1.75 -1.29 -2.78 to 0.20 0.07 -1.52 -2.86 to -0.18 <0.05

Lymphocytes Baseline 1.88 0.66 1.91 1.14 2.02 1.15

count/nl
Month 1 12 0.12 0.35 18 -0.30 0.42 <0.05 18 -0.13 0.70 -0.42 -0.72 to -0.11 <0.05 -0.24 -0.69 to 0.21

Month 3 12 0.34 0.47 <0.05 18 0.05 0.50 18 -0.09 0.64 -0.29 -0.66 to 0.08 -0.43 -0.87 to 0.01 <0.05

Month 6 12 0.31 0.51 <0.05 18 -0.19 0.83 18 -0.20 0.60 0.08 -0.50 -1.05 to 0.06 0.07 -0.51 -0.94 to -0.08 <0.01
Supplementary Table 5
Adverse events and safety parameters. Data are number of events (number of individuals). Control Diet
(CD), Fasting Mimicking Diet (FMD), Ketogenic Diet (KD).
CD FMD KD
Total adverse events 21 (11) 20 (14) 24 (14)
Respiratory tract infection 13 (9) 8 (7) 13 (12)
Transient reduced gait performance 0 6 (6) 0
Periapical periodontitis 1 (1) 1 (1) 0
Depression 0 1 (1) 0
Diarrhea 3 (3) 0 3 (3)
Dizziness 0 1 (1) 0
Feel cold 0 0 1 (6)
Headache 0 2 (2) 2 (2)
Nausea 0 0 2 (2)
Pain 4 (3) 1 (1) 2 (2)

Liver enzymes exceeding reference range 3fold 0 0 0

Total serious adverse events 1 (1) 3 (3) 2 (2)

Carpal tunnel syndrome 0 1 (1) 0
Ureteric colic 0 0 1 (6)
Lower urinary tract infection 1 (8) 2 (2) 1 (1)

Supplemental Table 6
Change from Baseline in Expanded Disability Severity Scale (EDSS).
Data are median (IQR). Mann-Whitney-U test for comparison between *CD
and FMD or **CD and KD. CD=Control Diet. FMD=Fasting Mimicking Diet.
KD=Ketogenic Diet.

CD FMD KD p* p**

Difference
0 (0 to 0) 0 (-1 to 0) 0 (-0.5 to 0) <0.05 <0.05
at month 3

Difference 0 (0 to -0.5 (-1.5 to

0 (-0.5 to 0.1) <0.05 <0.01
at month 6 0.5) 0)
Supplemental Table 7

Correlation analysis between baseline MS-54 scores and EDSS score of the 48 RRMS patients.
Deviations are given in brackets.

EDSS

rs p-value

Physical Health Composite (n=39) -0.624 <0,0001

Mental Health Composite (n=45) -0.255 0.09

Physical Function (n=47) -0.821 <0,0001

Health Perception (n=48) -0.242 0.1

Energy/Fatigue (n=47) -0.301 <0,05

Role Limitations Physical (n=46) -0.468 <0.001

Pain (n=48) -0.308 <0,05

Sexual Function (n=43) -0.105 0.5

Social Function (n=48) -0.32 <0,05

Health Distress (n=47) -0.104 0.5

Overall Quality of Life (n=48) -0.279 0.05

Emotional Well-Being (n=47) -0.111 0.5

Role Limitations Emotional (n=47) -0.143 0.3

Cognitive Function (n=47) -0.306 <0,05

Change in Health (n=48) -0.091 0.5

Satifaction with Sexual Function (n=46) -0.209 0.2

Supplementary Experimental Procedures
Fasting mimicking diet (Mouse).
On day 1, mice consume about 50% of their normal caloric intake (7.87 kJ/g). On days 2-3 mice
consume about 10% of their normal caloric intake (1.51 kJ/g). On average, mice consumed 11.07
kJ (plant-based protein 0.75 kJ, carbohydrate 5.32 kJ, fat 5 kJ) on each day of the FMD regimen.
Ketogenic Diet (Mouse).
The ketogenic diet was purchased from BioServ (F3666).
Cuprizone model.
C57BL/6 mice (5-weeks-old-female) were purchased from Charles River. Mice were fed 0.2%
w/w cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma) mixed into a ground standard
rodent chow (Harlan). Curprizone diet was maintained for 5 weeks; thereafter mice were put on
normal chow or FMD cycles (3 days of FMD followed by 4 days of regular chow) for another 5
weeks. 0, 1, 2, 3, 4 and 5 weeks after cuprizone withdrawal animals were euthanized. Brains
were perfused with 4% Paraformaldehyde (PFA), extracted, fixed in 4% PFA, paraffin-
embedded, sectioned and stained as described in the immunohistochemistry section.
EAE clinical disease severity score.
Clinical EAE was graded on a scale of 1-5 by established standard criteria as follows: score 0, no
observable disease; score 1, complete loss of tail tone; score 2, loss of righting; score 3, one hind
limb paralysis; score 4, both hind limbs paralysis; score 5, moribund/dead.
BrdU Injection.
BrdU (Sigma) was prepared in PBS (10 mg/mL stock solution) and intra peritoneal injected
according to experiment schedules. Two different BrdU injection schedules were conducted: For
oligodendrocyte precursor cells in the spinal cord, BrdU was injected 4 times daily (50 mg/kg).
For immune cell proliferation, 4 injections (1 mg/injection) was i.p. injected to mice 48 hours
prior to euthanasia.
In vitro T-cell assay.
Active EAE induced mice were sacrificed on Day 13 and spleens were removed. Blood was
collected for sera. Spleens were teased apart to single cell suspensions, RBC were lysed and
splenocytes were isolated by centrifugation. Cells were suspended in RPMI 1620 (Gibco)
supplemented with 10% fetal bovine serum and counted. Cells were plated at 2 x 105 cells per
well on a 96-well plate and treated with either 20 ug/ml MOG peptide or 10 ng/ml phorbol
myristate acetate (PMA) and 300 ng/ml ionomycin for 48 h to stimulate cytokine production.
Cytokine secretion was blocked during the last 5 h by treatment with monensin. FACS Analysis.
FACS analyses for different immune cell population were performed following standard
protocol. Freshly harvested splenocytes were stained with different immune cell markers (see
supplemental material and methods), followed by standard protocol for AnnexinV, intracellular
or BrdU staining. Analysis was performed with BD FACS diva on LSR II.
Statistical Analysis [Mouse study].
All data are expressed as the mean ± SEM. For mice, all statistical analyses were two-sided and
P values <0.05 were considered significant (* p<0.05, ** p<0.01, *** p<0.001). Differences
among groups were tested either by Student t-test comparison or one-way ANOVA followed by
Bonferroni post-test using GraphPad Prism v.5. Competing risk analysis was performed to assess
statistical differences in the rate of deaths.
Immunohistochemistry, antibodies and quantification.
Spinal cords were isolated from mice following standard protocols. Briefly, spinal cords were
fixed in 4% PFA overnight and stored in 0.5% sodium azide at 4 °C. Sectioning (5 µm) were
performed at the USC Histology Core. For paraffin embedded sections, the spinal cords were
fixed in 4% PFA, dehydrated in sequential concentrations of ethanol, cleared in xylene,
infiltrated, and then embedded with paraffin. Tissue was transversely cut in 9 µm sections.
Sections were de-paraffinized with xylene, and then hydrated with water. Antigen retrieval was
performed by placing sections in 0.1M citric acid pH 6, and boiled for 4 minutes in the
microwave. Slides were allowed to cool, coated with blocking solution (5% horse serum and
0.1% triton-X in PBS) for 1 hour at RT, stained with primary antibodies (See supplementary
material and methods) overnight at 4° C. For fresh frozen sections, the spinal cord sections were
air-dried and fixed in 4% PFA for 15 min, and washed with PBS containing 0.5% Tween-20.
Sections were permeabilized in 1% NP40 in PBS for 15 min, washed and blocked in 5% donkey
serum in 0.4% Triton for 1 hour, and incubated with various primary antibodies (See
supplementary material and methods) and incubated overnight in 4° C. Following day, sections
were washed, stained with secondary antibodies. Sections were washed with PBS, and covered
with Fluorshield Mounting Medium with DAPI (Vector Labs). Apoptotic cells were detected
using In Situ Cell Death Kit (Roche) following the manufacturer’s protocol. Images were
acquired and analyzed using a Nikon Eclipse 90i fluorescent and bright field microscope, along
with Metamorph 7.7 software. Images from the same spinal cord sections were “stitched” using
ImageJ Fiji. A minimum of 8 stitched spinal cord sections was quantified using ImageJ
(National Institute of Health). Raw images were digitally contrasted in the same way for each
stain within each time point. The digitally contrasted images were duplicated, and the region of
interest (ROI) was manually drawn on the image. Each image was thresholded equally for each
stain, and the region statistics tool was used to calculate the number of positive fluorescent pixels
located within the ROI. Antibodies used for Spinal cord immunostainings: anti-SMI32 (abcam,
ab28029, 1:1000), rabbit anti-MBP (zymed, 18-0038, 1:200), goat anti-GSTπ (Abcam, ab53943,
1:200), rabbit anti-NG2 (Millipore, AB5320, 1:100), rat anti-BrdU (Serotec, MCA2060, 1:200),
rat anti-CD4 (eBioscience, 14-0041-86, 1:100), rat anti-CD8 (abcam, ab22378, 1:100) and rat
anti-CD11b (Serotec, MCA711G, 1:200). Secondary antibodies include donkey anti-mouse
Alexafluor 647 (abcam, ab150103, 1:500) and donkey anti-rabbit Alexafluor 488 (abcam,
ab150069, 1:500).
Antibodies used for FACS analysis: CD3 Alexa700 (ebioscinece), CD4 PE-cy5 (ebioscinece),
CD44 APC (ebioscience), CD62L PE-cy7 (ebioscience), CD8 Alexa488 (ebioscience), CD11C
PE-cy7 (ebioscience), F4/80 PE-cy5 (ebioscience), CD25 PE-cy7 (ebioscience), FoxP3
Alexa488 (ebioscience), IL17 PE-cy7 (ebioscience) and IFNγ APC (ebioscience).
Demyelination Scoring.
Myelin was stained, using 9 μm sections, with solochrome cyanine as previously described
(Kiernan, 1984). Briefly, sections were stained for 90 minutes with Eriochrome Cyanine R
(Sigma; St. Louis, MO). Sections were washed in tap water, and then differentiated for 30 s in
10% iron (III) chloride (Sigma). Next, sections were counterstained with Van Gieson's stain for 2
min, washed, dehydrated in sequential concentrations of ethanol, cleared in xylene, and cover
slipped. Stained tissue was manually scored for the amount of demyelination on a scale from 0-5
(0=no demyelination, 1=one region of subpial white matter affected, 2=multiple regions of
subpial white matter affected, 3=multiple regions of white matter affected beyond subpial region,
4=parenchymal region affected, less than half of total white matter, 5=parenchymal region
affected, more than half of total white matter).
H&E Scoring.
Stained tissue was manually scored for the amount of inflammatory infiltration on a scale from
0-5 (0=no infiltration, 1=a few infiltrates in the leptomeninges, 2=organization of infiltrates
around blood vessels, 3=extensive perivascular cuffing with extension into the underlying
parenchyma, 4=infiltration in the parenchymal region, less than half of total white matter,
5=infiltration in the parenchymal region, more than half of total white matter).
Serum physiological biomarkers and cytokines.
Prior to blood collection, mice were withheld food for up to 4 hours. Serum was stored at -80°C.
β-hydroxybutyrate was measured with a colorimetric assay kit following the manufacturer’s
protocol (#700190, Cayman Chemical). IGF-1 (R&D), TNF-α (R&D), IFN-γ (R&D), and IL-17
(R&D) was measured following the manufacturer’s protocol.

Detailed Procedures for Clinical Trial.

Patients / Inclusion and Exclusion Criteria.
Patients were randomly allocated to I) KD for 6 months or II) 7 days FMD followed by
Mediterranean diet for 6 months or III) CD for 6 months. All patients met the following criteria:
Age > 18 < 67 years, stable disease modifying therapy (DMT) for at least six months prior to
inclusion, no DMT for at least six months or naive to therapy, EDSS < 6.5 and BMI > 18 < 45.
Exclusion criteria were primary or secondary progressive forms of MS, clinically relevant heart,
lung, liver, and kidney diseases, pregnancy or breast-feeding, other neurologic disorders, cancer,
weight loss therapy in the month prior to screening, relapse or steroid pulse therapy < 30 days prior
to screening, diabetes or other metabolic defects, bulimia, anorexia and drug abuse
Study Settings.
The study was approved by the local ethics committee and all participants gave informed written
consent according to the 1964 Declaration of Helsinki. RRMS patients fulfilling the current panel
criteria were prospectively recruited from our neurology outpatient clinic and via public
information all over Germany (Polman et al., 2005)
Interventions.
HRQOL in RRMS patients who met the inclusion criteria (n=60) were randomly assigned to three
study dietary interventions (n=20 per group): control diet (normal caloric standard diet), fasting
mimicking diet (FMD), and KD. To evaluate the food intake, a 115 item dietary self-record with
additional gaps for unlisted and individual foods or liquids and quantities prospectively over a
period of 7 days before baseline and between all other visits were recorded (Optidiet software
Version 5.1 GOE mbH, Büro Linden, Linden, Germany).
Modified human fasting mimicking diet.
A single cycle of the modified fasting mimicking diet consist of Day 1 – pre-fasting followed by
Day 2-8 – very low calorie diet. Day 1-prefasting consists of an 800 kcal (about 40% of normal
caloric intake similar to mouse Day1 FMD) monodiet (fruit, rice, or potatoes) by preference of
individuals. On the following day patients were recommended to use an oral laxative, Natrium
Sulfuricum (20-40 g). FMD consisted of 100 ml vegetable broth or vegetable juice with 1
tablespoon of linseed oil 3 times daily, plus additional calorie-free liquids. The daily calorie intake
was predefined with 200 – 350 kcal (10-18% of normal caloric intake similar to mouse Day 2-3
FMD). Patients were advised to drink 2-3 L of unsweetened fluids each day (water, and herbal
teas) and to use an enema if tolerated. After the 7-day fasting period solid foods were stepwise
reintroduced for three days, starting with a steamed apple at day 8. After the fasting and refeeding
period a normocaloric, plant-based Mediterranean diet was maintained until study end.
Low Glycemic Load Ketogenic Diet (KD).
Patients received KD for 6 months. Patients were recommended an average daily intake of < 50g
carbohydrates, > 160g fat and < 100g protein intake daily. Patients received detailed information
about nutritional facts, glycemic load and learned how to handle carbohydrates by an experienced
nutritional coach during group based workshops on 3 weekends. Details of the study specific KD
will be analyzed and published separately.
Control Diet.
Patients on CD met the criteria of a regular diet in German population as described in the “National
Nutrition Survey II” (http://www.was-esse-ich.de/index.php?id=44). We advised patients to stay
on their regular diet.
Assessments and Outcome Measures.
All participants were assessed for HRQOL at baseline, next visit occurred between days 3 to 17
after study outset (All FMD group members were assessed before breaking the FMD at day 8),
another visit was set after 3 months and the last regular visit proceeded after 6 months.
Primary Outcome Measures.
To measure HRQOL we used the generic and disease specific Multiple Sclerosis Quality of Life-
54 (MS-54) questionnaire consisting of 54 items. 2 composite scores concealing physical (PHCS)
and mental health (MHCS) are calculated from 12 directly obtained scales: Physical Health, Health
Perception, Energy/Fatigue, Role Limitations Physical, Pain, Sexual Function, Social Function,
Health Distress, Overall Quality of Life, Emotional Well Being, Role Limitations Emotional,
Cognitive Function. Additionally the MS-54 includes 2 single item scales: Change in Health and
Satisfaction with sexual functions. This instrument is validated for the German population, has
shown good reliability and validity within MS patients. We observed in our trial that patients with
a baseline EDSS score of > 2 < 3 compared with those in patients with baseline scores < 2 showed
> 5 points lower mean PHCS and MHCS scores per 0.5 EDSS incline. Thus we defined a change
of > 5 points to be clinically meaningful, which is in accordance with other studies (Rudick et al.,
2007, Michalsen and Li, 2013, Kappos et al., 2014)
Anthropometric Measurements.
Weight, fat and lean mass was specified by Air-Displacement Plethysmography (Bod Pod, Life
Measurements, Inc. Concord, CA), BMI was calculated as weight (kg)/height²(m). Seated blood
pressure was taken twice on each arm. All data were collected by trained study personnel only.
Depression.
The Beck depression inventory (BDI) was administered at each visit.
Fatigue.
Fatigue was measured with modified fatigue impact scale (MFIS), fatigue severity scale (FSS) and
visual analogue scale fatigue (VASF) at every visit.
Neurological Disability.
At baseline, month 3 and month 6, the EDSS(Kurtzke, 1983) and the Multiple Sclerosis Functional
Composite MSFC(Cutter et al., 1999) were performed to assess neurological disability. The
examiner was not blinded to treatment allocation.
Safety and other laboratory parameters.
Prior to blood collection all patients were on an overnight fast and samples were always taken at
the same time + 1h. Aspartate transaminase (AST), alanine transaminase (ALT), gamma-
glutamyltransferase (GT), WBCs, and lymphocytes were analyzed referring to international
standards at all visits. Other laboratory parameters as described in clinicaltrials.gov will be
analyzed and published separately.
Statistical Analysis [Human study].
Due to the small sample size of the pilot study, sufficiently powerful statistical analysis was not
possible. Baseline characteristics of the three intervention groups were compared using Kruskal-
Wallis tesw to determine uniformity of subjects. The primary end point, MS-54, and relevant
secondary endpoints were analyzed using non parametric Friedmann’s test for comparing intra
group differences at 3 or 4 time levels or Wilcoxon matched-pairs signed rank test at 2 time levels.
Next we performed baseline correction to increase the comparability between the groups. Thus we
calculated within each group the mean change from baseline (MCB) differences for every visit
and in a further step we compared MCB scores between all three groups using non parametric
Kruskal-Wallis test followed by Mann-Whitney-U test for pair wise comparisons. Associations
between variables were assessed using Spearman’s rank correlation coefficient (rs). All graphs are
based on mean and standard error of the mean (SEM) data. Because of the exploratory character
of the study analysis, alpha-adjusting was not performed. The test level for statistical significance
of differences between and within the treatment groups was defined as p = 0.05 (two-sided) for all
tests. For statistical analyses the following software was used: SPSS, version 20 (IBM, Armonk,
New York, US) and Graph Pad Prism, Version 5.0 (GraphPad Software, CA, US)
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