Science - USA (2022-06-10)

was 1 h after the food onset [Zeitgeiber time
(ZT) 1 h for CR-day-2h and ZT 13 h for the
CR-night-2h]. For the CR-spread group, the
phase was ZT12 because the mice ate equal
amounts during the day and night (fig. S4A).
The feeding pattern was also consistent with
daily changes in body weight (fig. S4B). With
the exception of the CR-spread group, in which
the food was equally distributed through-

out the day, body weight changed through-

out 24 h with a significant increase during the

feeding time (fig. S4B). This finding was more

pronounced in classic CR protocols, with the

highestbodyweightgainof3goccurring

between ZT0 and ZT4 in CR-day-2h mice

and between ZT12 and ZT16 in CR-night-2h

mice. These body weight gains are consist-

ent with the observation that mice eat their

entire allotment (2.7 to 3 g) as one single meal

within 2 h.

All groups maintained a normal nocturnal

locomotor activity pattern for life, with the

exception of the day-fed mice, which tended

to have more daytime activity (Fig. 1, C and D).

Overall, these long-term recordings showed

that when food was restricted to the daytime,

mice interrupted their“rest phase”to eat but

Acosta-Rodríguezet al., Science 376 , 1192–1202 (2022) 10 June 2022 2of11

Fig. 1. Effects of CR on body weight,

circadian behavior, and feeding in

C57BL/6J male mice.(A) Experimental

design showing feeding conditions for

each of the six groups. (B) Average body

weights (±SE) of mice in all six groups

(n = 43 for the AL group and n = 36 for

each of the CR groups) taken every 3 weeks

throughout the experiment. (C) Examples

of double-plotted actograms from each

experimental group overlaying wheel-

running (black histograms) and feeding

(red dots) behaviors. All mice were on AL

feeding for the first 6 weeks (period above

the line on the right of the actograms)

before the CR began. (D andE) Twenty-

four-hour profile of wheel-running activity

(D) and food intake (E) at different ages

(averaged over 21 days,n = 36 to 43 mice)

for each group. (F) Energy intake per

day (left axis) and number of food pellets

per day (right axis) for each group through-

out the experiment. For the AL group, the

dark line is average and the gray shading is

SE. All CR groups were limited to 70% of

AL consumption for the first 200 days of age

and were not adjusted after 200 days,

so no variation is observed. (G) Daily wheel-

running activity (average counts/min over

24 hours ± SE) throughout the experiment.

A

C CR-night-2h CR-day-2h CR-night-12h CR-day-12h CR-spread

DAY NIGHT DAY NIGHT DAY NIGHT

AL

DAY NIGHT DAY NIGHT DAY NIGHT

CR

(^2) AL
36
Age (months)
0 12 24 0 12 24 0 12 240 12 24 0 12 2401224
Time (h)
Feeding
Wheel-
Running
Activity
FGDaily Food Intake Daily Wheel-Running Activity
0
4
8
Food intake
(Pellets)
Age
(Months)
2
6
12
18
24
30
36
01224
0
40
80
Wheel activity (Count/min)
Age
(Months)
3
6
12
18
24
30
36
(^01224) Time (h)
D AL CR-night-2h CR-day-2h CR-night-12h CR-day-12h CR-spread
No Restriction
Time of day (h)
DAY NIGHT
Feeding groups 0 24^12
Unlimited amount of food
300mg food pellet (CR: 70% of intake)
Time of food access
Calories only CR-spread
(^) + Time CR-night-12h CR-day-12h
CR-night-2h
CR-day-2h
Calories
Calories

• Time
  (self-imposed)
  Body weight (g)
  20
  50
  211202935
  Age (months)
  B ALCR-night-12h
  CR-night-2h
  CR-day-12h
  CR-day-2h
  CR-spread
  E
  0
  10.8
  21.6
  0 200 400 600 800 1000 1200
  Days
  Energy intake (Kcal/day)^0
  10
  20
  30
  0 200 400 600 800 1000 1200
  Days
  Counts/min
  0
  10
  20 Food intake (pellets/day)
  RESEARCH | RESEARCH ARTICLE