Unloaded Minds - How Prolonged Unloading Shapes Human Performance in Lunar Gravity (UnloadedMinds)

During spaceflight and lunar missions, reduced gravitational loading causes the muscles and nervous system to decondition rapidly. While muscle wasting is well documented, much less is known about how the brain and spinal cord adapt to the absence of normal mechanical loading - and how these central changes interact with muscle deterioration to impair movement control.

This study investigates how two weeks of simulated lower limb unloading affects the brain's movement programs (motor engrams), the functional connection between nerves and muscles, and overall movement quality. Healthy participants undergo 14 days of Unilateral Lower Limb Suspension (ULLS), a model in which one leg is kept unloaded using a raised shoe and crutches while the other leg functions normally. After the unloading period, participants travel to Bordeaux, France, where they walk on a treadmill for the first time - in lunar gravity - during a parabolic flight.

Before and after the unloading period, participants are assessed with MRI (muscle size), force plate tests (jump performance and balance), nerve stimulation (number of functional motor units), and treadmill gait analysis combining brain activity (EEG), muscle activity (EMG), joint movement, and plantar pressure measurements. The same gait analysis is repeated during the parabolic flight under lunar gravity (0.16G).

The results will shed light on how the brain and muscles adapt to disuse and reactivate under partial gravity - with direct relevance to astronaut safety and rehabilitation, as well as clinical conditions involving prolonged limb immobilization.

Study Overview

• Status: Recruiting
• Conditions: Unilateral Lower Limb Suspension (Simulated Microgravity)
• Intervention / Treatment: Behavioral: Unilateral lower limb suspension

Detailed Description

The study combines two spaceflight analogues in sequence: a 14-day Unilateral Lower Limb Suspension (ULLS) protocol followed by a parabolic flight campaign simulating lunar gravity (0.16G). This design allows investigation of both the neuromuscular consequences of unloading and the acute reactivation of the system upon first weight-bearing locomotion under partial gravity.

During ULLS, participants keep one lower limb (randomized in a counterbalanced manner) unloaded continuously using a shoe with a 10-cm thick sole and short-length crutches with forearm support. The suspended limb remains in a near-straight position and swings freely from the hip during all upright and ambulatory activity. Compliance is verified daily via telephone interviews, continuous midcalf skin temperature monitoring, and accelerometer data. Fragmin 5000 IU is administered subcutaneously once daily throughout the ULLS period as thromboprophylaxis.

Following the 14-day ULLS period, participants travel to Bordeaux where they continue the ULLS protocol until initiating the first steps on a treadmill during the lunar gravity phases of a parabolic flight. Each participant is allocated 15 parabolas (~20 seconds of 0.16G each): parabolas 1-8 at 3 km/h and parabolas 9-15 at 5 km/h.

Gait analysis is performed on a treadmill at 3 and 5 km/h at baseline (pre-ULLS) and during the parabolic flight. Kinematic data (knee and ankle joint angles) are recorded bilaterally using 2D electronic goniometers (Biometrics, 500 Hz) in the frontal and sagittal planes. Ground reaction forces and spatiotemporal parameters (step and stride length, cadence, step width, gait cycle duration and variability) are recorded using plantar pressure insoles (200 Hz). All signals are synchronized to allow left/right (loaded/unloaded) differentiation.

EEG is recorded during all gait sessions using a 64-channel cap (EasyCap GmbH) with electrodes placed per the international 10-20 system (reference: FCz; ground: AFz). Data are processed in BrainVision Analyzer 2.2 using visual inspection, automated artefact rejection (gradient threshold >60 µV), ICA, and bandpass filtering (0.5-40 Hz). Cortical current density in the motor cortex (M1, Brodmann area 4) is computed using the sLORETA module.

Surface EMG is recorded continuously during gait from eight bilateral lower limb muscles (rectus femoris, lateral hamstrings, gastrocnemius lateralis, tibialis anterior) using a wireless system (Delsys, USA; 2000 Hz), with electrode placement per SENIAM guidelines. Data are bandpass filtered (12-500 Hz), full-wave rectified, and lowpass filtered (7 Hz) to derive linear activation envelopes.

Motor Unit Number Estimation (MUNE) is performed using the MScanFit method, which applies incremental electrical stimuli to the peripheral nerve at 0.6-second intervals across varying intensities, recording the full stimulus-response curve via surface EMG to estimate the number of functional motor units innervating lower leg muscles.

MRI is performed before and after ULLS using a 3-Tesla system. T2-weighted images are acquired to quantify thigh and calf muscle volumes bilaterally. Participants rest supine for ~30 minutes prior to each scan to minimize fluid shift artefacts.

Neuromuscular performance and balance are assessed before ULLS and immediately after the parabolic flight using dual force plates (MuscleLab, 200 Hz). Measures include countermovement jump height, peak power, rate of force development, and centre-of-pressure-based postural stability variables.

Statistical Analysis Data will be assessed for normality using the Shapiro-Wilk test. Continuous outcome variables will be analysed using linear mixed-effects models (LMMs) with Time (pre, post), Leg (unloaded, control), and their interaction (Time × Leg) as fixed effects, and Subject as a random intercept. This approach appropriately handles the repeated-measures, within-subject structure of the data and accommodates missing observations without listwise deletion. Degrees of freedom will be estimated using the Satterthwaite approximation. Where a significant interaction is detected, pairwise post-hoc comparisons will be performed with Bonferroni correction. Effect sizes will be reported as Cohen's d.

For the parabolic flight gait analysis, outcomes recorded at 1G (pre-ULLS) and 0.16G (during flight) will be compared using LMMs with Gravity Condition (1G, 0.16G), Leg (unloaded, control), and Speed (3 km/h, 5 km/h) as fixed effects, and Subject as a random intercept.

Associations between central (EEG-derived cortical current density, MUNE) and peripheral (muscle volume, jump performance, EMG) outcomes will be examined using Pearson or Spearman correlation coefficients, depending on data distribution.

A sample size of 12 participants provides 95% power to detect a moderate effect size (f = 0.6) at a significance level of α = 0.05 for a within-subject design comparing outcomes across gravity conditions (G*Power; λ = 17.3, critical F = 4.85). Twenty-four participants will be recruited to account for potential dropouts (including motion sickness during the parabolic flight, which may be a common, impossible-to-predict cause of dropout) and ensure the required sample is available for analysis. Statistical analyses will be performed in R (R Foundation for Statistical Computing). The significance threshold is set at p < 0.05.

• Study Type: Interventional
• Enrollment (Estimated): 24
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Rodrigo Fernandez Gonzalo, PhD, Research group leader; Phone Number: +46852483514; Email: rodrigo.fernandez-gonzalo@ki.se
• Study Contact Backup: Name: Mirko Mandic, PhD; Phone Number: +46700823625; Email: mirko.mandic@ki.se

Study Locations

• Sweden
  • Stockholm County
    • Huddinge, Stockholm County, Sweden, 14152
      • Recruiting
      • Karolinska Institutet
      • Contact: Lars Frelin, Facility manager; Phone Number: +46852483632; Email: lars.frelin@ki.se

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

- 18-60 years of age, willing to participate

Exclusion Criteria:

• high activity level according to the GLTEQ
• contraindications to exercise
• pregnancy
• contraindications to MRI
• active cardiovascular disease
• cerebrovascular disease including previous stroke
• aneurysm (large vessels or intracranial)
• pulmonary disease including pulmonary hypertension or COPD
• metabolic diseases (including: hyper/hypoparathyroidism, hyper/hypothyroidism, Cushing's disease, type 1 or type 2 diabetes)
• active inflammatory bowel disease
• kidney disease
• malignancy
• recent steroid treatment (within the past 6 months), or hormone replacement therapy
• coagulation disorders
• musculoskeletal or neurological diseases
• diseases requiring long-term medication, including statin therapy
• smoking

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm

Intervention / Treatment

Experimental: ULLS; There is only one arm, where one leg is the experimental, unloaded leg, and the other one is the control (loaded) leg

Behavioral: Unilateral lower limb suspension; Unilateral Lower Limb Suspension (ULLS) is a validated spaceflight analog for the lower limbs, which mimics unloading specifically in the lower limbs 3. In ULLS, participants wear a shoe with ~100 mm elevated sole and use short-length crutches aided by a hand grip and forearm support distal to the elbow. This allows the contralateral lower limb to remain unloaded in a straight position and move passively at the hip. The ULLS model specifically targets skeletal muscle unloading in the lower limb with no or minor systemic deconditioning, and it imposes minimal restrictions on participants' daily activities. A key advantage of ULLS is that the contralateral, weight-bearing limb provides a valid inherent control accounting for individual factors such as nutrition, genetics, and lifestyle.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Muscle volume	Three days before the initiation of the ULLS intervention, and at day 15 of unloading, all participants will undergo an MRI scan to assess thigh and calf muscle size. Muscle volume will be evaluated using cross-sectional T2-weighted images acquired on a 3-Tesla MR system. Anatomical landmarks and standardized procedures will ensure that the same segment is scanned at both time points. Participants will rest in a supine position for approximately 30 minutes prior to each scan to minimize measurement interference caused by fluid shift. Images will be analysed using manual segmentation to delineate individual muscle boundaries in each cross-sectional slice; muscle volume is then calculated by summing the cross-sectional areas of the target muscles across all slices and multiplying by slice thickness.	Three days before the initiation of the ULLS intervention and at day 15 of unloading, for both thigh and calf muscles bilaterally.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Gait analysis	Gait analysis will be performed before the ULLS phase and during the lunar gravity phases of a parabolic flight at the two selected gait speeds of 3 km/h and 5 km/h. Kinematic parameters such as knee and ankle joint angles are recorded using 2-dimensional electronic goniometers (Biometrics). Kinematic data will be recorded in the frontal and sagittal plane at a sampling frequency of 500 Hz. Kinetic and spatiotemporal parameters like the force distribution are recorded using pressure insoles with a sampling frequency of 200 Hz. Spatiotemporal parameters are crucial indicators of gait stability and overall mobility. They include variables such as step length, stride length, cadence, step width, and gait cycle duration and their variability.	Gait analysis will be performed three-to-five days before the ULLS phase and during the lunar gravity phases of a parabolic flight
Electroencephalogram	The data is recorded using a 64-channel electroencephalography (EEG) cap adapted to the size of the head. The electrodes are arranged in the international 10-20 system around the motor cortex. Electrode FCz is used as a reference, AFz as ground. The EEG cap, which is fixed with a chin strap to prevent displacement, is permeable to air to ensure appropriate air ventilation during exercise. The distances between the electrodes are approx. 5 cm to prevent a possible false signal due to salt bridges caused by perspiration. Each electrode is filled with SuperViscTM electrode gel (EasyCap GmbH, Herrsching, Germany) for signal transmission. The use of active EEG electrodes developed by Brain Products (Gilching, Germany) allows the recording of electrocortical activity during exercise without movement artefacts.	Three-to-five days before the ULLS phase and during the lunar gravity phases of a parabolic flight, during the gait analysis tests.
Electromyography	Surface EMG activity will be continuously recorded for the following 8 muscles of the left and right lower limbs: rectus femoris (RFL and RFR), lateral hamstrings (HL and HR), gastrocnemius lateralis (GASL and GASR), and tibialis anterior (TAL and TAR). These muscles are activated by the Central Nervous System (CNS) to produce the gait. The EMG signals will be collected using a commercial surface wireless EMG system (Delsys, USA).	Three-to-five days before the ULLS phase and during the lunar gravity phases of a parabolic flight, during the gait analysis tests.
Muscle function and balance	Muscle function will be assessed by a standardized countermovement jump on force plates (MuscleLab, Norway). Balance will be assessed by a sway test where participants will balance on one leg with their eyes closed and eyes open, this is also done on force plates.	Three days before initiation of ULLS and immediately after the parabolic flight.
Motor unit number estimation	Motor unit number estimation (MUNE) test that assesses how many motor units are functioning in a muscle. It starts with placing surface electrodes on the skin to record electrical activity from the target muscle. A mild electrical stimulus is applied to the nerve supplying the muscle, and the response (motor unit potential) is measured. By gradually increasing the stimulus and analyzing the size and number of responses, an estimate of the total number of motor units is calculated.	Three days before initiation of ULLS and immediately after the parabolic flight.

Collaborators and Investigators

• Sponsor: Karolinska Institutet
• Collaborators: German Sport University, Cologne

Publications and helpful links

General Publications

• Badali C, Wollseiffen P, Schneider S. Shades of gravity - effects of planetary gravity levels on electrocortical activity and neurocognitive performance. Brain Struct Funct. 2024 Jun;229(5):1265-1277. doi: 10.1007/s00429-024-02803-6. Epub 2024 May 3.
• Badali C, Wollseiffen P, Puck L, Klein T, Schneider S. Neurophysiological markers of cognitive workload under altered gravity conditions using a gamified dual-task paradigm. Sci Rep. 2026 Jan 9;16(1):1292. doi: 10.1038/s41598-025-34426-0.
• Fernandez-Gonzalo R, Irimia JM, Cusso R, Gustafsson T, Linne A, Tesch PA. Flywheel resistance exercise to maintain muscle oxidative potential during unloading. Aviat Space Environ Med. 2014 Jul;85(7):694-9. doi: 10.3357/asem.3856.2014.
• Tesch PA, Lundberg TR, Fernandez-Gonzalo R. Unilateral lower limb suspension: From subject selection to "omic" responses. J Appl Physiol (1985). 2016 May 15;120(10):1207-14. doi: 10.1152/japplphysiol.01052.2015. Epub 2016 Feb 4.
• Berg HE, Dudley GA, Haggmark T, Ohlsen H, Tesch PA. Effects of lower limb unloading on skeletal muscle mass and function in humans. J Appl Physiol (1985). 1991 Apr;70(4):1882-5. doi: 10.1152/jappl.1991.70.4.1882.
• Berg HE, Tesch PA. Changes in muscle function in response to 10 days of lower limb unloading in humans. Acta Physiol Scand. 1996 May;157(1):63-70. doi: 10.1046/j.1365-201X.1996.476217000.x.

Study record dates

Study Major Dates

• Study Start (Actual): May 1, 2026
• Primary Completion (Estimated): May 31, 2028
• Study Completion (Estimated): March 31, 2029

Study Registration Dates

• First Submitted: May 13, 2026
• First Submitted That Met QC Criteria: May 20, 2026
• First Posted (Actual): May 26, 2026

Study Record Updates

• Last Update Posted (Actual): May 26, 2026
• Last Update Submitted That Met QC Criteria: May 20, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: Gait analysis; Skeletal muscle; Unilateral lower limb suspension; Motor engrams; Space physiology
• Other Study ID Numbers: 2025-02981-01 - UnloadedMinds

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No