This was a one-year, single-blinded study, involving a treatment group and a non-treated group of children and adults with ASD. Single-blind means that the clinical evaluators were blinded, but the participants were not. The reason for this design is that we wanted to conduct a long-term study on the effects of a comprehensive set of dietary and nutritional treatments, similar to what the Autism Research Institute has recommended for years [ 72 ]. We believe that length of time is too long for a double-blind, placebo-controlled format, because too many participants would drop out. Also, it is extremely difficult to do a double-blind study when making major diet changes, especially the switch to a “healthy” diet as described below. A single-blind study is still a rigorous study design for assessments for which the clinical observer is fully blinded, and the additional data from unblinded participants provides some useful information similar to an open-label study. Since this was an exploratory new treatment, the choice of sample size was roughly estimated based on previous studies. The authors confirm that all ongoing and related trials for this drug/intervention are registered (Clinicialtrials.gov: NCT02059577).

The study was advertised by email to approximately 2500 ASD families in Arizona, using the contact list of the Autism Society of Greater Phoenix and the Autism/Asperger’s Research Program at Arizona State University (ASU). Interested ASD families attended a one-hour informational meeting, and consenting families joined the study. Neurotypical families were recruited from friends of the ASD families and professionals who work with ASD families. Participants were recruited for the study from October 2011 to April 2014. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board (IRB) of Arizona State University.

No major changes in behavioral or medical treatments in the previous two months, and no intention to make such changes during the 12 months of the study.

Note that there was no exclusion for individuals with ASD with specific metabolic or genetic disorders. No participants reported unusual genetic or metabolic disorders, but there was no attempt to screen for those, and it is likely that some may have existed.

The characteristics of the study participants are listed in Table 1 . The ASD treatment group, ASD non-treatment group, and the neurotypical controls have similar age distributions (mostly children, some teens, and a few adults), and similar gender distributions (mostly male). The ASD treatment and non-treatment group have similar diagnoses (mostly autism). For the ASD group, 100% met the criteria for ASD per the CARS-2, and 88% met the criteria for ASD per the ADOS (most of the 8 participants who met only the CARS-2 criteria were high-functioning teens/adults who in the clinical judgement of the evaluator were clearly on the ASD spectrum, so they were admitted to the study).

After enrollment and ADOS/CARS-2/Reynolds Intellectual Assessment Scales (RIAS)/Severity of Autism Scale (SAS-Pro) assessment, participants were randomly assigned to either the Treatment or Non-treatment group. The study coordinator enrolled participants, conducted the randomization, and assigned participants to interventions. She was not involved in any of the evaluations. The Treatment group began treatment immediately, whereas the Non-treatment group was asked not to make any changes in medical, nutritional, therapy, or education treatment for 12 months. The Non-treatment group was promised that they would receive all the supplements and diet advice at the end of the study if they made no major changes to any educational interventions for 12 months, which helped minimize the drop-out rate. Participants were enrolled on a rolling basis, and participants with similar ages were matched and then randomly assigned to one of the two groups.

Vitamins and carnitine were measured at UC-San Diego by some of our team using liquid chromatography tandem mass spectrometry (LC-MS/MS) (SCIEX, Redwood City, CA, USA) as previously described with modifications [ 73 ] (PMID 25705365). The absolute concentrations of acylcarnitines were calculated using stable isotope internal standards. The levels of vitamin and co-factors were normalized using neurotypical control baseline values and reported as peak area ratios.

Some testing was done by LabCorp, and some by Doctor’s Data. Both commercial laboratories are approved by the Clinical Laboratory Improvement Amendments (CLIA) program operated by the US Department of Health and Human Services which oversees approximately 200,000 laboratories in the US. Samples were taken by courier from our medical office to the local LabCorp testing facility in Phoenix (refrigerated or on dry ice as appropriate). Samples for Doctor’s Data were shipped overnight, either with cold packs or on dry ice as appropriate.

Biomarkers in blood and urine were measured at the beginning and end of the study in the children and adults in the ASD groups, and one time at the start of the study in the neurotypical group. The samples were sent in a blinded fashion to the laboratories for testing.

Handgrip strength, an indicator of muscle strength, was assessed by using a pneumatic, adjustable squeeze pinch-gauge/dynamometer (Baseline Evaluation Instruments; White Plains, NY, USA) by a study nurse unaware of the treatment status of the subject. This instrument is a reliable and valid method for obtaining muscle force measurements in children and adults, and takes only a few min. One of three bulb sizes was used, depending on the participant’s hand size, and the same size was used at beginning and end of the study. Each participant was shown how to squeeze the bulb with one hand, and then three measurements were taken, and the highest value was recorded.

2.10. Autism Severity and Overall Functioning Assessments

A large number of assessments of autism severity and overall functioning were used because we hypothesized that there might be improvements in many different areas. Most assessments were conducted for the autism treatment and non-treatment groups at the beginning and end of the study. The PGI-2 was assessed at month, 3, 6, 9, and 12 for the treatment group, and at month 12 for the non-treatment group. The initial assessments were conducted before randomization, so neither the participants nor the evaluator knew which group they were in. At the end of the study, the evaluator first conducted the ADOS and RIAS assessments in a blinded manner. The evaluator then conducted the CARS-2 and SAS-Pro assessments, which did involve some discussion with the parents (except for a few high-functioning participants who did not have a parent available). The Vineland was conducted by phone by a different blinded evaluator. The blinding of the evaluators at the beginning and end of the study was complete in all cases except for one CARS-2/SAS-Pro assessment at the end of the study (a participant inadvertently commented about the treatment).

Autism Diagnostic Observation Schedule (ADOS): The ADOS is a 1-h structured interaction and is one of the primary tools used for clinical diagnosis of autism and autism spectrum disorders. It involved a blinded evaluation by clinicians certified in ADOS assessment.

Reynolds Intellectual Assessment Scales (RIAS): The RIAS assesses verbal and non-verbal IQ and memory. It involves a 20–30 min blinded assessment by clinicians using a variety of standardized IQ and memory activities.

Childhood Autism Rating Scale 2 (CARS-2): The CARS-2 evaluation was conducted after the ADOS and RIAS evaluations, and was based partly on the participant’s performance and interactions on those assessments, and partly on specific interactions and interview questions with the participant and their parents. The clinician was blinded as to the participant’s treatment status, but the participants and the parents were not, so this is classified as “semi-blinded”.

Severity of Autism Scale (SAS-Pro): The SAS-Pro is a single number on a scale of 0–10 to evaluate overall severity of autism symptoms. It was evaluated by the professional evaluator after the ADOS, RIAS, and CARS-2, so it was classified as “semi-blinded”.

All of the ADOS, RIAS, CARS-2, and SAS-Pro evaluations were done by the same professional evaluator at beginning and end (either EP or JI).

Vineland Adaptive Behavior Scale II (VABS-II): The VABS-II was conducted by a phone interview with the participant’s parents (or the participants in a few cases for high-functioning adults), so it was classified as “semi-blinded”. One evaluator (RLA) conducted all the interviews at beginning and end. The calculated raw scores were then converted into an age equivalent. However, most of the questions are geared towards younger ages, and there are fewer questions for the older ages, and a difference of one point for the older participants can result in a jump of more than 1 year of developmental age. Therefore, for scoring purposes we set a maximum age for the following subscales, based on the age at which questions became sparse: Receptive—11 years; Communication—12.3 years; Written—15.3 years; Domestic—15.3 years; Play—19 years; Coping—17.8 years; Gross Motor—6.8 years; Fine Motor—6.8 years; the other subscales (Personal and Community) had a maximum of 22 years. Only 2 participants in the treatment group and 2 participants in the non-treatment group had some scores at the maximum of any subscale, except for the Gross Motor and Fine Motor subscales, which had 32% and 44% of the treatment and non-treatment group scoring at the maximum for those subscales. So, although scores for Gross Motor and Fine Motor skills are reported, they need to be interpreted cautiously.

Parents (or the participants in a few cases for high-functioning adults) completed an initial medical history form, and at the beginning and end of the study they also completed several questionnaires to assess autism and related symptoms, including the following: ATEC, Pervasive Developmental Disorders Behavior Inventory (PDD-BI), Social Responsiveness Scale (SRS), 6-item Gastrointestinal Severity Index (6-GSI), Short Sensory Profile (SSP), Aberrant Behavior Checklist (ABC), and Medical History. Also, the Parent Global Impressions-2 (PGI-2) was completed at the end of months 3, 6, 9, and 12, to assess changes during the previous 3 months.