A Randomized Controlled Trial on the Effects of a 12-Week High- vs. Low-Intensity Exercise Intervention on Hippocampal Structure and Function in Healthy, Young Adults

Physical exercise affects hippocampal structure and function, but the underlying neural mechanisms and the effects of exercise intensity remain incompletely understood. Therefore, we undertook a comprehensive, multi-modal 3T and 7T MRI randomized controlled trial (Netherlands Trial Register - NL5847) in which we randomized 52 young, non-athletic volunteers to a 12-week low- or high-intensity exercise program. Using state-of-the-art methods, we investigated changes in hippocampal volume, as well as changes in vasculature, neuro-metabolites, and peripheral growth factors as potential underpinnings. Cardiorespiratory fitness improved over time (p < 0.001), but no interaction with exercise intensity was found (p = 0.48). Accordingly, we did not observe significant interactions between exercise condition and time on MRI measures (all p > 0.06). However, we found a significant decrease in right hippocampal volume (p < 0.01), an increase in left hippocampal glutathione (p < 0.01), and a decrease of left hippocampal cerebral blood volume (p = 0.01) over time, regardless of exercise condition. Additional exploratory analyses showed that changes in brain-derived neurotrophic factor (p = 0.01), insulin-like growth-factor (p = 0.03), and dorsal anterior cingulate cortex N-acetyl-aspartate levels (p = 0.01) were positively associated with cardiorespiratory fitness changes. Furthermore, a trend toward a positive association of fitness and gray-matter cerebral blood flow (p = 0.06) was found. Our results do not provide evidence for differential effects between high-intensity (aerobic) and low-intensity (toning) exercise on hippocampal structure and function in young adults. However, we show small but significant effects of exercise on hippocampal volume, neurometabolism and vasculature across exercise conditions. Moreover, our exploratory results suggest that exercise might not specifically only benefit hippocampal structure and function, but rather has a more widespread effect. These findings suggest that, in agreement with previous MRI studies demonstrating moderate to strong effects in elderly and diseased populations, but none to only mild effects in young healthy cohorts, the benefits of exercise on the studied brain measures may be age-dependent and restorative rather than stimulatory. Our study highlights the importance of a multi-modal, whole-brain approach to assess macroscopic and microscopic changes underlying exercise-induced brain changes, to better understand the role of exercise as a potential non-pharmacological intervention.


. Sample Size Calculations
We aimed to investigate the neural mechanisms underlying brain volume changes resulting from exercise interventions. First, we determined the sample size to assess a hypothesized effect on neurometabolite signaling. A previous study investigating exercise effects on anterior cingulate cortex glutamate concentration changes reported an effect size of d=1.48 (Cohen d) in young participants (Maddock et al., 2016). As this study used an acute exercise intervention, we expected a more moderate effect size for a longitudinal exercise intervention of d=1.0. To detect the effect of exercise, with a power of 80% and an α of 0.05, we would need 12 participants per exercise intervention group. Taking a 20% dropout into account (due to MRI data quality), we would need at least 15 subjects in each exercise intervention group. Secondly, we calculated the required sample size to assess the effect of exercise on vascular outcome measures. A previous study using contrast-enhanced MRI assessed the effect of a dietary intervention with flavanol on CBV in the hippocampus (Brickman et al., 2014). This study detected an increase in CBV with an effect size of d=0.75. We expect the dietary intervention to have effects of a similar magnitude as the exercise intervention. With an effect size of d=0.75, a power of 80%, and an α of 0.05, we would need 16 subjects to detect a difference before and after the exercise intervention. Taking a 20% dropout into account, we would need at least 20 subjects in each exercise group. Low-Intensity Group: • BBB -The class consists of a short warm-up followed by various exercises that focus on the belly, buttocks, and legs.
• Body power* -This is a group workout to music using simple and effective exercises with barbells and dumbbells. It is focused on the muscular endurance of the whole body.
• Essentrics -At Essentrics you get an effective toning workout to music using dynamic stretches and fluid movements without using gear. The main goals are a slimmer silhouette, more flexibility, and better posture.
• Pilates -In this class the focus is on: posture and control, flexibility, breathing, and awareness. In Pilates, you do floor exercises that target all postural muscles in the body, especially the abdominal and back muscles. You do the exercises slowly, fluently and in collaboration with your breath. You concentrate on doing the exercises carefully and accurately, and not on the number of repetitions. The result is better posture and flexibility.
• TrippleShape barre workout -Ballet Barre workout is a combination of ballet, pilates, and yoga to contemporary music.
• Yoga basic -This is a basic yoga class. The focus lies on: stretching and holding postures.
• Kinesis -Kinesis Training takes traditional exercises (like chest press, lat pull, row) and combines them with functional movements (like reaching, squatting, bending). It develops balance, core/overall strength, and flexibility for people of all fitness levels.
• Abs -15 minutes of abs training using own-body weight.
• Basic movement -In this class good technique of the most basic exercises using own-body weight is being practiced. This lesson focuses on questions such as: How do I perform the squat correctly? What is a pull-up and how do I do it? What variations are there for a deadlift?
• Mobility class -This lesson focuses on increasing flexibility in muscles and joints.
• Calisthenics -This is a class to practice exercises like a handstand, muscle up, human flag, front and back lever.
• Core*-This lesson focuses on posture and strengthening the abdominal and back muscles in a controlled manner. The training does not only consist of floor exercises but is also offered in challenging circuit forms.
• W.A.C.* -Weightlifting Aerobic Circuit: this workout is originally based on Olympic weightlifting, a valuable full-body workout.
*For Core, W.A.C., and body power, participants were only allowed to do a maximum of 1 of these 3 per week for this sports program.

High-Intensity Group:
• Combat -In this class you train your entire body during circuit training. It includes techniques from (kick) boxing, self-defense techniques, strength, and conditioning to increase your heart rate.
• Fitness training -This is circuit training in which all available materials are used. Walking and jumping are used a lot in this training, the intensity is largely determined by the participants themselves. A high-intensity training involving both strength and condition.
• Fit Fight -This is a powerful cardio workout inspired by Eastern martial arts such as karate, boxing, taekwondo, and Muay Thai to music.
• Kick & shape -This is an intensive class in which participants learn various punching and kicking techniques against the punching bag to music.
• Spinning -In this class participants sit on a spinning bike, on which, under the guidance of the teacher, they go on a 'bike tour' in which different speeds alternate. The resistance is chosen by the participants themselves.

• Step & dance -
Step & dance is a choreographically challenging, advanced step class, with the goal of increasing aerobic fitness.
• Total body workout -This class is a short piece of aerobics hi/low impact after a warm-up. This is followed by exercises for all muscle groups: back, arms, abdomen, shoulders, chest, and legs. This class improves the participant's overall fitness, both cardio and muscle endurance are trained.
• Synrgy -Synrgy is a large device, with rods, ropes, bells, and whistles. Participants can follow group training sessions of 30 minutes several times a day, in which they do high-intensity interval training in circuit form.
• H.I.I.T. -High-Intensity Interval Training consists of short periods of intense effort, followed by short recovery moments. Participants train their general condition but also strength and speed, using simple materials or body weight.
• Crossfit -This small group training is based on the principles of a fitness concept from the USA: functionality, variation, and high intensity. Participants train speed, (muscle) endurance, strength, flexibility, and coordination.

Details about the exercise intervention:
Exercise progression was monitored by tracking their sports center visits using an automated fingerprint entrance system and by using weekly questionnaires on exercise duration and activities. Additionally, participants received an HR monitor (Polar, Finland) to measure HR during each training session. In case the regularity of participants' training sessions was decreasing, participants were contacted and motivated to continue exercising by one of the experimenters. No home-program or non-exercise components were added to the intervention. No adverse events occurred during the exercise intervention in this study. Multiple comparison corrections using Sidak's resulted in an α*=0.02. Model selection was based on an adjusted top-down procedure, in which the resulting models were compared using the Bayesian information criterion (BIC); the model was consequently tested using chi-square (χ²) tests.

Supplementary
Supplementary Table 3 | Exploratory analyses testing the association of all variables with VO2max, and hippocampal or dACC volume change were conducted using linear models in R, including the baseline measure of the explanatory variables, VO2max, and sex as covariates (α=0.05).
Supplementary Table 4 | Mean and standard deviation for pre-and post-exercise intervention per variable.

Supplementary Figure 2 | Exploratory analysis -associations with VO2max:
Significant associations of the change in power output during the VO2max test, NAA concentration in the dACC, blood BDNF levels, and blood IGF levels with change in VO2max.

Physical activity besides the intervention:
Minutes spent on physical activity did not change for either low-or high-intensity condition from pre to post (low: t(37)=0.26, p=0.79; high: t(36)=-0.12, p=0.91). When splitting these hours up into different categories we also did not find any changes from pre to post intervention in: minutes spent walking (low: t (43)